1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile

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:

From 10 JULY 2019 to 22 AUGUST 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

The range of temperature was under the desired range. The stock solution vas autoclaved for 20 minutes. The preculture was prepared 5 days before the test start. All these deviations met the validation criteria and were considered non-critical deviations.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

The concentrations to be tested are based on non GLP pre-test. Replicates: 6 replicates for blank control, 6 replicates for solvent control, 3 replicates for each treatment. Duration: 72 hours, Lighting 4440-8880 lux, Temperature 20.3-23.6ºC

Details on sampling:

- Concentrations: 0.1 µg/L nominal concentration
- Sampling method:
- Sample storage conditions before analysis:

Vehicle:

no

Remarks:

The solubility of the test item in test medium is < 0.1 µg/L (see validation report VB18062903G926). Therefore, this study was performed as a limit test at 0.1 µg/L to show that there is no toxicity at the LOQ (limit of quantification) of the analytical m

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: 86.81
- Source (laboratory, culture collection): MBM Sciencebridge GmbH (Intitut für Pflanzenphysiologie of Universität Göttingen)
- Age of inoculum (at test initiation): 2016
- Method of cultivation: permanetn culture on solid agar at 2-8ºC

ACCLIMATION
- Acclimation period: 96h
- Culturing media and conditions (same as test or not): nutrient medium and incubated
- Any deformed or abnormal cells observed: none

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

The test vessels were filled with 45 ± 1 mL of the respective test solution and incubated
open (covered with perforated plastic foil acting as a stopper) for 72 hours, shaken on an
orbital shaker to keep the algae in suspension.

Test temperature:

20.3 – 23.6 °C

pH:

Blank control 0h -7.1 72h-8.5
Solvent control 0h-7.1 72h-8.7
Test item 0.1 µg/L 0h-7.1 72h-8.6

Nominal and measured concentrations:

0.1 µg/L nominal concentration
0.1 µg/L measured concentration

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass flasks total volume 65 mL
- Type (delete if not applicable): open (covered with perforated plastic foil acting as a stopper)
- Material, size, headspace, fill volume: 45 ± 1 mL of the respective test solution
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- Initial cells density: 2.4 *10^3 cells/mL
- Control end cells density:
- No. of organisms per vessel: BLANK CONTROL 2380 CN/mL (0h), 10510 CN/mL (24h), 88660 CN/mL (48h), 439893 CN/mL (72h); SOLVENT CONTROL 2380 CN/mL (0h), 10457 CN/mL (24h), 83270 CN/mL (48h), 388733 CN/mL (72h), TEST ITEM 2380 CN/mL (0h), 10753 CN/mL (24h), 82847 CN/mL (48h), 412993 CN/mL (72h)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates):6
- No. of vessels per vehicle control (replicates):6

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:72h
- Light intensity and quality: 4440 – 8880 lux
- Salinity (for marine algae):

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

TEST CONCENTRATIONS
- Spacing factor for test concentrations: according pre-test
- Justification for using less concentrations than requested by guideline: poor solubility of the test item
- Range finding study
- Test concentrations: 0.1 µg/L measured concentration
- Results used to determine the conditions for the definitive study: 0.1 µg/L

Reference substance (positive control):

yes

Remarks:

The 72h-EC50 values of potassium dichromate were determined in a separate reference test. For the estimation of the 72h-EC50 values of the positive control, the fits showed sufficient statistical correspondence of the data with the dose-response-equation.

Key result

Duration:

72 h

Dose descriptor:

other:

Remarks:

72h EyC50

Effect conc.:

> 0.1 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Determinable

Remarks:

The concentration of test substance which results in a 50 percent reduction in growth rate (ErC50) relative to the control within 72hrs exposure. It is regarded as acute endpoint.

Key result

Duration:

72 h

Dose descriptor:

other:

Remarks:

72h ErC50

Effect conc.:

> 0.1 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Determinable

Remarks:

Yield inhibition

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 0.1 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other:

Remarks:

Yield

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 0.1 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.1 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other:

Remarks:

Yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.1 µg/L

Nominal / measured:

nominal

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): none
- Unusual cell shape: none
- Colour differences: none
- Flocculation: none
- Adherence to test vessels: none
- Aggregation of algal cells: none
- Other:
- Any stimulation of growth found in any treatment: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium: none

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50:
- Other:
ErC50 = 0.66 mg/L
EyC50 = 0.40 mg/L

Reported statistics and error estimates:

Mean and Standard Deviation of Cell Number/mL

6.2 Positive Control
Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a separate reference test.
6.2.1 Biological Results of the Reference Study (201901R301)
The results of the last study with the positive control K2Cr2O7 are presented in the following
table. The study was performed under GLP conditions in February 2019.
Table 6.2-a Biological Results of K2Cr2O7

Parameter	Value	95% confidence interval
72h ErC50	0.66 mg/L	0.64 – 0.69 mg/L
72h E yC50	0.40 mg/L	0.38 – 0.41 mg/L

 

Validity criteria fulfilled:

yes

Conclusions:

One valid experiment was performed.
The solubility of the test item in test medium is < 0.1 µg/L (see validation report
VB18062903G926). Therefore, this study was performed as a limit test at 0.1 µg/L to show
that there is no toxicity at the LOQ (limit of quantification) of the analytical method. Acetone was used to spike the test solution. A stock solution containing 1 mg/L in acetone was
prepared. For preparing the treatment 100 µL/L stock solution was used.
For the solvent control, acetone in a concentration of 100 µL/L was used.
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 yield2 were determined from the
cell number at the respective observation times.
In comparison to the blank control, a slight effect can be seen with the solvent control.
Therefore, the results of the treatment are related to the solvent control.
Since no inhibition of algae growth has occurred, no statistical evaluation is carried out.
At the beginning and at the end of the test, the content of the test item in the test solutions
was determined using LC-MS-MS–determination. At the beginning of the test, the test item
could be detected very well. At the end of the test no test item could be detected. Therefore the results are related to the nominal values.
The 72h-EC50 values of potassium dichromate were determined in a separate reference
test. For the estimation of the 72h-EC50 values of the positive control, the fits showed sufficient statistical correspondence of the data with the dose-response-equation. The values
were within the range of the laboratory.
The pH of the blank control should not fluctuate by more than 1.5 units. The change was
1.4 units in the blank control.
All validity criteria were met.
No observations were made which might cause doubts concerning the validity of the study
outcome. The result of the test can be considered valid.

Description of key information

According to OECD 201 resp. EU c.3

The solubility of the test item in test medium is < 0.1 µg/L (see validation report
VB18062903G926). Therefore, this study was performed as a limit test at 0.1 µg/L to show that there is no toxicity at the proposed LOQ (limit of quantification) of the analytical method. Acetone was used to spike the test solution. A stock solution containing 1 mg/L in acetone was prepared. For preparing the treatment 100 µL/L stock solution was used.
For the solvent control, acetone in a concentration of 100 µL/L was used.
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 yield1 were determined from the cell number at the respective observation times.
In comparison to the blank control, a slight inhibition can be seen with the solvent control.
Therefore, the results of the treatment are related to the solvent control.
Slight stimulation of algal growth could be observed in the treatment compared to the solvent control. The test item showed no toxicity. Since no inhibition of algae growth has occurred, no statistical evaluation was carried out.
At the beginning and at the end of the test, the content of the test item in the test solutions was determined using LC-MS-MS–determination. At the beginning of the test, the test item could be detected very well. At the end of the test no test item could be detected. Therefore the results are related to the nominal values

Key value for chemical safety assessment

EC50 for freshwater algae:

0.1 µg/L

EC10 or NOEC for freshwater algae:

0.1 µg/L