p-[(5-cyano-1,6-dihydro-2-hydroxy-1,4-dimethyl-6-oxo-3-pyridyl)azo]-N-(2-ethylhexyl)benzamide

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:

from 2017-07-16 to 2017-07-20, with the definitive exposure phase from 2017-07-17 to 2017-07-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

p-[(5-cyano-1,6-dihydro-2-hydroxy-1,4-dimethyl-6-oxo-3-pyridyl)azo]-N-(2-ethylhexyl)benzamide

EC Number:

250-205-6

EC Name:

p-[(5-cyano-1,6-dihydro-2-hydroxy-1,4-dimethyl-6-oxo-3-pyridyl)azo]-N-(2-ethylhexyl)benzamide

Cas Number:

30449-81-1

Molecular formula:

C23H29N5O3

IUPAC Name:

p-[(5-cyano-1,6-dihydro-2-hydroxy-1,4-dimethyl-6-oxo-3-pyridyl)azo]-N-(2-ethylhexyl)benzamide

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
The test concentration of Disperse Yellow 198 was analytically verified via HPLC-DAD in the saturated solution and the control at the start of the exposure (0 hours). At the start of the exposure (0 hours), sampling was carried out after preparation of the loading level. At the end of the exposure (72 hours), no analytical monitoring was carried out, because a measured test item concentration above LOD was not observed at the start of the exposure.

Test solutions

Vehicle:

no

Details on test solutions:

Preparation of the Saturated solution
A saturated solution with a nominal loading of 10.0 mg test item/L was prepared once 24 hours 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 dilution water. The saturated solution was stirred for 24 hours (1100 rpm at room temperature) 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 were 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. The following filtrate, i.e. the saturated solution, was used as a limit concentration in the test. During filtration, the filter was always be kept covered.
The solution was checked via laser beam (Tyndall effect), which was negative.

Test loading
All terms related to concentration level are given as loading level because partly dissolved compounds and mixtures cannot be related to concentrations. One limit concentration were tested with a nominal loading of 10.0 mg/L. The limit loading is based on the results of a non-GLP preliminary range finding test.

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

Test organisms

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): 3 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.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No

Test conditions

Hardness:

Not specified

Test temperature:

min.: 22.0 max.: 23.0 mean value: 22.5

pH:

Nominal test item loading pH-values
[mg/L] Start; 0 hours End; 72 hours
10.0 7.70 8.33
Control 7.91 8.75

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

Nominal: 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 100 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 10^3 cells/mL, Actual: mean 6839 cells/mL
- Control end cells density: Mean 1914404 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 0.064a2EDTA 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: 5771 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. No self-fluorescence was found in the preliminary range finding test at the saturated solution.

Reference substance (positive control):

yes

Remarks:

(Potassium dichromate)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

other: NOEL

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

other: LOEL

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

other: EL10/20/50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

other: NOEL

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 5

Duration:

72 h

Dose descriptor:

other: LOEL

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 6

Duration:

72 h

Dose descriptor:

other: EL10/20/50

Effect conc.:

> 10 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 saturated solution was visually clear throughout the exposure period.
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

The toxicity of potassium dichromate (SIGMA ALDRICH, 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 2017-04-18 to 2017-04-21. 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 0.559 0.754 ± 0.518
95% confidence interval 0.538 – 0.581
Yield inhibition
EyC50 0.320 0.398 ± 0.291
95% confidence interval 0.242 – 0.341
SD = Standard deviation

Reported statistics and error estimates:

EL-values and statistical analyses
EL-values of the growth rate and yield inhibition were estimated empirically based on the results of the single treatment level (Limit test design).

NOEL and LOEL values
NOEL/LOEL were determined by calculation of statistical significance of growth rate and yield. As a standard, a t-test was used for NOEL/LOEL calculations. When running a t-test a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normal distribution of data. The Levene median test was used for equal variance. 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.

Any other information on results incl. tables

Cell Densities

 

Nominal test item loading		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 hours		24 hours		48 hours		72 hours
10.0		1		6839		28483		275569		1958850
		2		6839		26712		243687		1964643
		3		6839		26753		230976		1818226
		4		6839		31830		289836		2091163
		5		6839		28234		283563		1915018
		6		6839		31350		346261		2317694
		Mean		6839		28894		278315		2010932
Control		1		6839		29477		289637		2021642
		2		6839		35584		296296		2274155
		3		6839		35084		318803		2065707
		4		6839		26818		248006		1828409
		5		6839		21039		232404		1709203
		6		6839		25404		174996		1587306
		Mean		6839		28901		260024		1914404

 

 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		Growth rate inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
10.0	1		1.89	-1		1952011	-2
	2		1.89	-1		1957804	-3
	3		1.86	1		1811387	5
	4		1.91	-2		2084324	-9
	5		1.88	0		1908179	0
	6		1.94	-4		2310855	-21
	Mean	(-)	1.89	-1	(-)	2004093	-5
Control	1		1.90			2014803
	2		1.94			2267316
	3		1.90			2058868
	4		1.86			1821570
	5		1.84			1702364
	6		1.82			1580467
	Mean		1.88			1907565

 

 

 

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	1.46	2.29	1.94	1.90	0.414	21.8	22.4
	2	1.65	2.12	2.04	1.94	0.251	13.0
	3	1.64	2.21	1.87	1.90	0.288	15.1
	4	1.37	2.22	2.00	1.86	0.445	23.9
	5	1.12	2.40	2.00	1.84	0.653	35.5
	6	1.31	1.93	2.21	1.82	0.457	25.2
				Mean	1.87
				SD ±	0.04
				CV [%]	2.36

 

SD = Standard deviation CV = Coefficient of variation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, Disperse Yellow 198 was found not to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours.
Effect values were determined as follows (based on nominal test item loading):
The EL50-values for inhibition of growth rate (ErL50) and yield (EyL50) after 72 hours were > 10.0 mg/L, respectively. The NOEL-values for both inhibition of growth rate and yield after 72 hours were 10.0 mg/L, respectively.

Executive summary:

The toxicity of Disperse Yellow 198 to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3 from 2017-07-16 to 2017-07-20, with the definitive exposure phase from 2017-07-17 to 2017-07-20 at the test facility. The aim of the study was the determination of NOEL, LOEL, EL₁₀, EL₂₀- and EL₅₀-values of growth rate and yield over a period of 72 hours.

 

The study was conducted under static conditions with an initial cell density of 6839 cells/mL. A saturated solution with a nominal loading of 10.0 mg test item/L was prepared with dilution water 24±1 hour prior to the start of the exposure. An appropriate amount of the test item was weighed out. The saturated solution was stirred for 24 ±1hour (1100 rpm) with a magnetic stirrer at room temperature. 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 negative.

Six replicates were tested for the limit loading level and six for the control. The test media were colourless and clear throughout the test period. The environmental conditions were within the acceptable limits.

The measured concentration of the test item was even < LOD at the start of the exposure (0 hours), therefore no analysis at the end of the exposure was necessary.

 

All effect values given are based on the nominal test item loading of Disperse Yellow 198.

 

NOEL, LOEL, EL_x-values of Disperse Yellow 198 (0 - 72 hours)

                  based on the nominal test item loadings

	Growth Rate Inhibition nominal test item loading [mg/L]
NOEL	10.0
LOEL	> 10.0
ErL10	> 10.0
ErL20	> 10.0
ErL50	> 10.0
	Yield Inhibition nominal test item loading [mg/L]
NOEL	10.0
LOEL	> 10.0
EyL10	> 10.0
EyL20	> 10.0
EyL50	> 10.0