Reaction mass of 6-amino-2-anilino-4-methyl-5-{(E)-[4-nitro-2-(substituted)phenyl]diazenyl}nicotinonitrile and 2-amino-6-anilino-4-methyl-5-{(E)-[4-nitro-2-(substituted)phenyl]diazenyl}heteromonocyclic nitrile

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:

31 October 2011 to 03 November 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000.

Deviations:

no

Principles of method if other than guideline:

none

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Stability in water: Not indicated
Solubility in water: ≤24.5 ng/L at 20 °C (determined in NOTOX project 497887)

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. In addition, the filter was kept for possible analysis of undissolved residue.

Frequency: at t=0 h, t=24 h and t=72 h
Volume: 1 ml
Storage: Samples were stored in a freezer until analysis.

At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.

Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at the highest substance concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Additionally, singular reserve samples of 1 ml were taken from all test solutions for possible analysis.

Details on test solutions:

The batch of FAT 41043/A tested was a red powder, solid with a purity of 99.8 % and consisted of two isomeric forms. The substance was not completely soluble in test medium at the loading rate initially prepared.

Preparation of test solutions started with a loading rate of 100 mg/l applying a 15-minute period of magnetic stirring combined with a treatment with ultrasonic waves. The obtained aqueous mixture was subsequently magnetically stirred for a period of two days to ensure maximum dissolution in test medium and subsequently filtered through a 0.45 µm membrane filter (rc55, Whatman).The lower test concentrations were prepared by subsequent dilutions of the filtrate in test medium. The final test solutions were all clear and colourless.

After preparation, volumes of 50 ml were added to each replicate of the respective test concentration. Subsequently, 1 ml of an algal suspension was added to each replicate providing a cell density of
10E4 cells/ml.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata,
- Strain: strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture.
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

-

Post exposure observation period:

no

Hardness:

0.24 mmol/l (24 mg CaCO3/l)

Test temperature:

21.9 °C - 22.9 °C

pH:

7.9-8.3

Dissolved oxygen:

not measured

Salinity:

not measured

Nominal and measured concentrations:

Nominal concentrations: 0.1, 1.0, 10 and 100 % of a 0.45 µm filtered solution prepared at a loading rate of 100 mg/l.

Measured concentration: The actual concentration measured in the undiluted filtrate at the start of the test was 35 ng/l. This concentration dropped to 40 % of initial at the end of the test. Note that the measured concentrations were only indicative. However, the concentration measured at the start of the exposure was higher that the water solubility of FAT 41043/A.

Similar results were obtained for the undiluted filtrate incubated without algae.

Based on these results the Time Weight Average (TWA) concentration was calculated to be 21 ng/l.

Details on test conditions:

TEST SYSTEM

- Test duration: 72 hours
- Test type: Static
- Test vessels: 100 ml, all-glass, containing 50 ml of test solution
- Medium: M2
- Cell density: An initial cell density of 1 x 10E4 cells/ml.
- Illumination: Continuously using TLD-lamps of the type ‘Cool-white’ of 30 Watt, with a light intensity within the range of 77 to 88 uE.m-2.s-1.
- Incubation: Capped vessels were distributed at random in the incubator and as such were daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.
- Type (delete if not applicable): closed
- Aeration: no
- Control end cells density: 1783000 cells/ml
- No. of vessels per concentration (replicates): 6 for the highest concentration, 3 for the lower concentrations, 1 replicate of each concentration without algae
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometer

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

21 ng/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: -

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 21 ng/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: -

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 21 ng/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: -

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

21 ng/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: -

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 21 ng/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: -

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 21 ng/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: -

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no

No significant differences were recorded between the values for growth rate or yield at any of the test concentrations when compared to the control group.

Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.

Results with reference substance (positive control):

Potassium dichromate reduced growth rate of this fresh water algae species at nominal concentrations of 0.56 mg/l and higher.

The EC50 for growth rate reduction (ERC50: 0-72h) was 1.5 mg/l with a 95% confidence interval ranging from 1.1 to 2.1 mg/l. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/l. Hence, the ERC50: 0-72h for the algal culture tested corresponds with this range.

The EC50 for yield inhibition (EYC50: 0-72h) was 0.52 mg/l with a 95% confidence interval ranging from 0.34 to 0.78 mg/l. The historical ranges of the 72h-EC50 for yield inhibition lie between 0.43 and 1.1 mg/l. Hence, the EYC50: 0-72h for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. Statistical analysis of the data was not needed as the effects recorded were not significant (<10 %). No EC50-values could be calculated because the test substance proved to be non-toxic.

 

Table1           Mean cell densities (x 10⁴cells/ml)

FAT 41043/A % of filtrate prep. at 100 mg/l	Exposure time (hours)
	0	24	48	72
control	1.0	9.2	42.8	178.3
0.10	1.0	8.8	41.3	164.6
1.0	1.0	9.1	39.5	170.2
10	1.0	9.1	40.3	167.1
100 (21)	1.0	9.2	40.3	164.3

() – between brackets TWA concentration is given [ng/l]

 

 

Table2           Percentage reduction of growth rate (total test period) and percentage inhibition of yield

FAT 41043/A % of filtrate prep. at 100 mg/l	Mean growth rate		Yield (0-72 h)
	µ (0-72 h)	Reduction (%)	x10^4cells/ml	Inhibition (%)
control	0.07198		177.29
0.10	0.07085	1.6	163.64	7.7
1.0	0.07135	0.9	169.24	4.5
10	0.07106	1.3	166.05	6.3
100 (21)	0.07083	1.6	163.26	7.9

() – between brackets TWA concentration is given [ng/l]

 

 

Table3           Percentage reduction of growth rate at different time intervals

FAT 41043/A % of filtrate prep. at 100 mg/l	Mean growth rate
	µ (0-24 h)	Reduction (%)	µ (24-48 h)	Reduction (%)	µ (48-72 h)	Reduction (%)
control	0.09230		0.06412		0.05954
0.10	0.09054	1.9	0.06439	-0.4	0.05762	3.2
1.0	0.09178	0.6	0.06141	4.2	0.06084	-2.2
10	0.09171	0.6	0.06221	3.0	0.05927	0.4
100 (21)	0.09265	-0.4	0.06135	4.3	0.05850	1.7

() – between brackets TWA concentration is given [ng/l]

Validity criteria fulfilled:

yes

Conclusions:

No reduction of growth rate or inhibition of yield was recorded at any of the concentrations of FAT 41043/A tested. The EC50 for growth rate reduction (ERC50: 0-72h) and the EC50 for yield inhibition (EYC50: 0-72h) were beyond the range tested, i.e. exceeded a TWA concentration of 21 ng/l obtained in an undiluted filtrate prepared at a loading rate of 100 mg/l. The NOEC for growth rate reduction and yield inhibition was 21 ng/l. Due to the very low solubility of FAT 41043/A in water, concentration levels that might be toxic for algae could not be reached.

Description of key information

The toxicity to aquatic algae and cyanobacteria of FAT 41043/A was investigated according to OECD Guideline Nr. 201, EU Method C.3 and GLP principles. No reduction of growth rate was recorded at any of the concentrations of FAT 41043/A tested.
The EC50 for growth rate reduction (ERC50: 0-72h) was beyond the range tested, i.e. exceeded a TWA concentration of 21 ng/l obtained in an undiluted filtrate prepared at a loading rate of 100 mg/l. The NOEC for growth rate reduction was 21 ng/l.

Additional information

Due to the very low solubility of FAT 41043/A in water, concentration levels that might be toxic for algae could not be reached.