2-[N-(2-cyanoethyl)-4-[(2,6-dichloro-4-nitrophenyl)azo]anilino]ethyl acetate

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 2016-11-14 to 2017-03-03, with the definitive exposure phase from 2017-02-28 to 2017-03-03

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:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Principles of method if other than guideline:

The method was modified according to OECD series no. 23. The test substance solution was orange. Since this may affect the light intensities for the algae cultures the volumes of test media in the test vessels was lowered in order to minimize the water layer and the path length.

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Determination of the test substance:
The test concentration was analytically verified via HPLC-DAD in the saturated solution, the dilutions and the control at the start of the exposure (0 h) and at the end of the exposure (72 h).

Sampling for the analytical monitoring:
At the start of the exposure (0 h), sampling was carried out after preparation of the loading levels. Samples of the aged media were taken from the test vessels (with algae) at the end of the exposure (72 h).

Vehicle:

no

Details on test solutions:

Preparation of the Saturated solution
A saturated solution with a nominal loading of 100 mg test item/L was prepared once 24 ± 1 hour prior to the start of the exposure. An appropriate amount of the test item was weighed out. The test item was applied onto a glass slide. The glass slide with the test item was inserted into a glass bottle with an appropriate amount of demineralized water. The saturated solution was stirred for 24 hours (1100 rpm) with a magnetic stirrer. Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL). The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL of the filtrate will be discarded. The filtration was interrupted for 10 minutes to allow adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. During filtration, the filter will always be kept covered.
The solution was checked via laser beam (Tyndall effect), which was positive. Then the components of the dilution water were added to the saturated solution.

Test loading
All terms related to concentration level are given as loading level because partly dissolved compounds and mixtures cannot be related to concentrations. The saturated solution and four dilution levels out of the saturated solution were tested in a geometrical series with a dilution factor of 2: 6.25 - 12.5 - 25.0 - 50.0 - 100% of the saturated solution.

Control
Six replicates (without test item) were incubated under the same conditions as the test loadings.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDÁK
- Strain: SAG 61.81
- Source (laboratory, culture collection): SAG, Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 2567 – 5130 lux corresponding to 35 - 70 µE ∙ m-2 ∙ s-1 for 24 h per day.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Hardness:

Not specified

Test temperature:

min: 22°C max: 22.5°C mean value: 22.3°C

pH:

Nominal test substance loading pH-values
[% of the saturated solution] Start; 0 h End; 72 h
100 7.57 7.45
50.0 7.61 7.50
25.0 7.69 7.56
12.5 7.78 7.67
6.25 7.89 7.91
Control 8.03 8.27

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

Nominal: 6.25 - 12.5 - 25.0 - 50.0 - 100% of the saturated solution
Measured: < LOQ

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 25 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: Nominal: approximately 2 - 5 x 10E+3 cells/mL, Actual: mean 6839 cells/mL
- Control end cells density: Mean 1619065 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Composition of Dilution Water
according to the guideline
Component Concentration [mg/L]
NH4Cl 15
MgCl2 x 6 H2O 12
CaCl2 x 2 H2O 18
MgSO4 x 7 H2O 15
KH2PO4 1.6
FeCl3 x 6 H2O 0.064
Na2EDTA x 2 H2O 0.1
H3BO3 0.185
MnCl2 x 4 H2O 0.415
ZnCl2 3 x 10^-3
Na2MoO4 x 2 H2O 7 x 10^-3
CoCl2 x 6 H2O 1.5 x 10^-3
CuCl2 x 2 H2O 1 x 10^-5
NaHCO3 50
pH 8.1 ± 0.2



OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120
µE ∙ m-2 ∙ s-1, mean value: 5684 lux,



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
-Chlorophyll a-fluorescence
The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. Self-fluorescence was observed in the range finding test. A separate replicate for the loading levels were prepared without algae and treated the same as the other test replicates. The self-fluorescence of each measuring point was taken into account in the evaluation.

Reference substance (positive control):

yes

Remarks:

(Potassium dichromate)

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

71.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: CI: 51.1 - 96.9

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: The test item concentrations were clear during the exposure. The algae in the saturated solution were agglutinated after 72 hours. No agglutination of the algae was observed in the control replicates.
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

The toxicity of potassium dichromate (SIGMA, batch number MKBV0900V, purity 99.0 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2016-10-11 to 2016-10-14 (with headspace). The reference item toxicity is in the valid range following test facility SOPS.

EC50-Values of the Reference Item
based on nominal concentrations mg/L, (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Growth Rate inhibition
ErC50 0.435 0.764 ± 0.513
95 % confidence interval 0.414 – 0.458
Yield inhibition
EyC50 0.230 0.402 ± 0.293
95 % confidence interval 0.179 – 0.322
SD = Standard deviation

Reported statistics and error estimates:

EL-values and statistical analyses
EL-values of growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression.

NOEL, LOEL and analyses
The NOEL and LOEL were determined by calculation of statistically significant differences of growth rates and yield. As a standard, One Way Analysis of Variance (ANOVA) and DUNNETT’s test were used for NOEL/LOEL calculations. When running a One Way Analysis of Variance, a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.

Software
The data for the tables in this report were computer-generated and have been rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures.
Calculations were carried out using software
- Excel, MICROSOFT CORPORATION
- SigmaPlot, SPSS INC.
- GraphPad Prism, GRAPHPAD SOFTWARE, INC.

 Cell Densities

Nominal test item loading		Replicate		Cell density [cells/mL]
[% of the saturated solution]		No.		0 hours		24 hours		48 hours		72 hours
100		1		6839		18997		124160		1275845
		2		6839		17694		118694		1194005
		3		6839		15939		105047		1009960
		Mean		6839		17543		115967		1159937
50.0		1		6839		23116		177682		1701315
		2		6839		24181		178045		1740933
		3		6839		21686		166716		1575672
		Mean		6839		22994		174148		1672640
25.0		1		6839		26314		200072		1983674
		2		6839		24006		184664		1926675
		3		6839		19991		146959		1537108
		Mean		6839		23437		177232		1815819
12.5		1		6839		32304		237478		1834565
		2		6839		24214		180292		1877870
		3		6839		22710		173510		1767560
		Mean		6839		26409		197093		1826665
6.25		1		6839		28330		181211		1871081
		2		6839		26209		200856		2021595
		3		6839		21893		164480		1655494
		Mean		6839		25477		182182		1849390
Control		1		6839		26502		153724		1319882
		2		6839		23649		177413		1796293
		3		6839		28260		181211		1564846
		4		6839		22979		101758		1538512
		5		6839		25413		162227		1746785
		6		6839		22789		185998		1748072
		Mean		6839		24932		160389		1619065

 

 

 Evaluation after 72 hours

             Statistically significant differences of growth rates and yield compared to
                  control values are marked (+), not significant differences are marked (-).

 

Nominal test item loading	Replicate	Growth rate		Inhibition of growth rate	Yield		Inhibition of yield
[% of the saturated solution]	No.	[d^-1]		[%]	[cells/mL]		[%]
100	1		1.74	4		1269006	21
	2		1.72	5		1187166	26
	3		1.67	9		1003121	38
	Mean	(+)	1.71	6	(+)	1153098	28
50.0	1		1.84	-1		1694476	-5
	2		1.85	-1		1734094	-8
	3		1.81	0		1568833	3
	Mean	(-)	1.83	-1	(-)	1665801	-3
25.0	1		1.89	-4		1976835	-23
	2		1.88	-3		1919836	-19
	3		1.81	1		1530269	5
	Mean	(-)	1.86	-2	(-)	1808980	-12
12.5	1		1.86	-2		1827726	-13
	2		1.87	-3		1871031	-16
	3		1.85	-2		1760721	-9
	Mean	(-)	1.86	-2	(-)	1819826	-13
6.25	1		1.87	-3		1864242	-16
	2		1.90	-4		2014756	-25
	3		1.83	-1		1648655	-2
	Mean	(-)	1.87	-2	(-)	1842551	-14
Control	1		1.75			1313043
	2		1.86			1789454
	3		1.81			1558007
	4		1.81			1531673
	5		1.85			1739946
	6		1.85			1741233
	Mean		1.82			1612226

 

 Section-by-Section and Average Specific Growth Ratesof the Control Group
(0 - 72 hours)

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 hours)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.35	1.76	2.15	1.75	0.398	22.7	29.4
	2	1.24	2.02	2.32	1.86	0.554	29.9
	3	1.42	1.86	2.16	1.81	0.371	20.5
	4	1.21	1.49	2.72	1.81	0.801	44.3
	5	1.31	1.85	2.38	1.85	0.532	28.8
	6	1.20	2.10	2.24	1.85	0.562	30.4
				Mean	1.82
				SD ±	0.04
				CV [%]	2.13

 

SD = Standard deviation CV = Coefficient of variation

Validity criteria fulfilled:

yes

Conclusions:

Under the test conditions, the test substance was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 h with the following effect values (based on nominal loading levels): EL50 values with 95% confidence intervals for both inhibition of growth rate (ErL50) and yield (EyL50) after 72 h were > 100%, respectively.

Executive summary:

The toxicity of the test substance to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the OECD Guideline 201 and EU Method C.3. The study was conducted under static conditions with an initial cell density of 6839 cells/mL. A saturated solution with a nominal loading of 100 mg test substance/L was prepared with demineralized water 24±1 h prior to the start of the exposure. An appropriate amount of the test substance was weighed out. The saturated solution was stirred for 24 ±1 h (1100 rpm) with a magnetic stirrer. Undissolved particles were removed by membrane filtration. The following filtrate, i.e. the saturated solution was used in the test. The solution was checked via laser beam (Tyndall effect), which was positive. Thereafter the components of the dilution water were added to the saturated solution. The saturated solution and four dilution levels out of the saturated solution were tested in the definitive test in a geometrical series with a dilution factor of 2:6.25 - 12.5 - 25.0 - 50.0 - 100% of the saturated solution. Three replicates were tested for each concentration and six replicates for the control. The test media were clear throughout the test period. The environmental conditions were within the acceptable limits. The concentration of the test substance was analytically verified via HPLC‑DAD at the start of the exposure of the saturated solution, the dilutions and the control. The recovery rates of the test substance at test start were below the lowest calibration level (LCL). Therefore, all effect values given are based on the nominal test substance loading levels. Under the study conditions, the test substance was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 h. However, the % inhibition of growth rate at the highest loading level was only 6% (not significant) and % inhibition of yield was 28%. The following effect values (based on nominal loading levels) were determined based on the results: The 72 h EL50 with 95% confidence intervals for both inhibition of growth rate (ErL50) and yield (EyL50) after 72 h were > 100% (i.e. 100 mg/L) respectively (Volker, 2017).

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

Additional information

The toxicity of the test substance to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the OECD Guideline 201 and EU Method C.3. The study was conducted under static conditions with an initial cell density of 6839 cells/mL. A saturated solution with a nominal loading of 100 mg test substance/L was prepared with demineralized water 24±1 h prior to the start of the exposure. An appropriate amount of the test substance was weighed out. The saturated solution was stirred for 24 ±1 h (1100 rpm) with a magnetic stirrer. Undissolved particles were removed by membrane filtration. The following filtrate, i.e. the saturated solution was used in the test. The solution was checked via laser beam (Tyndall effect), which was positive. Thereafter the components of the dilution water were added to the saturated solution. The saturated solution and four dilution levels out of the saturated solution were tested in the definitive test in a geometrical series with a dilution factor of 2:6.25 - 12.5 - 25.0 - 50.0 - 100% of the saturated solution. Three replicates were tested for each concentration and six replicates for the control. The test media were clear throughout the test period. The environmental conditions were within the acceptable limits. The concentration of the test substance was analytically verified via HPLC‑DAD at the start of the exposure of the saturated solution, the dilutions and the control. The recovery rates of the test substance at test start were below the lowest calibration level (LCL). Therefore, all effect values given are based on the nominal test substance loading levels. Under the study conditions,the test substance was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 h. However, the % inhibition of growth rate at the highest loading level was only 6% (not significant) and % inhibition of yield was 28%. The following effect values (based on nominal loading levels) were determined based on the results: 72 h EL50 with 95% confidence intervals for both inhibition of growth rate (ErL50) and yield (EyL50) after 72 h were > 100% (i.e. 100 mg/L) respectively (Volker, 2017).