3-(diethylamino)-7-oxo-7H-[1]benzopyrano[3',2':3,4]pyrido[1,2-a]benzimidazole-6-carbonitrile

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 2018-04-16 to 2018-04-20, with the definitive exposure phase from 2018-04-17 to 2018-04-20

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

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:

3-(5-chlorobenzoxazol-2-yl)-7-(diethylamino)-2-benzopyrone

EC Number:

252-722-2

EC Name:

3-(5-chlorobenzoxazol-2-yl)-7-(diethylamino)-2-benzopyrone

Cas Number:

35773-43-4

Molecular formula:

C20H17ClN2O3

IUPAC Name:

3-(5-chloro-1,3-benzoxazol-2-yl)-7-(diethylamino)-2H-chromen-2-one

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
The test item concentrations of Disperse Yellow 184.1 and the control were analytically verified via HPLC-DAD 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 levels with algae. The measured test item concentrations were below the LCL for all test item loading levels. Therefore, at the end of the exposure (72 hours), no analytical monitoring was carried out. The samples were analysed with an HPLC-DAD method, implemented under non-GLP and documented in the GLP raw data. The method was not validated, since the measured concentrations were below the lowest calibration level.

Test solutions

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 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, 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 15 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). The Tyndall effect 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. 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. The concentrations are 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 item replicates.

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): 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.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

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
[% of the saturated solution] Start; 0 hours End; 72 hours
100 7.66 7.94
50.0 7.64 7.96
25.0 7.64 7.94
12.5 7.68 8.03
6.25 7.82 8.34
Control 8.00 8.60

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

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 5523 cells/mL
- Control end cells density: Mean 1871402 cells/mL
- No. of vessels per concentration (replicates): 3
- 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: 6297 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 definitive test in 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.:

100 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate und yield

Effect concentrations 2

Duration:

72 h

Dose descriptor:

other: LOEL

Effect conc.:

> 100 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 other: % saturated solution

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 media 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 2018-04-09 to 2018-04-12. 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 0.574 0.752 ± 0.554
95% confidence interval 0.549 – 0.596
Yield inhibition
EyC50 0.337 0.399 ± 0.294
95% confidence interval 0.313 – 0.345
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 not calculated. Effect values of growth rates were determined to be below 10% inhibition, so a determination of ErL 10 / 20 / 50 was considered as not possible.
Yield inhibition showed one effect value >10% inhibition in the lowest tested test item concentration. Since the EyL 10 / 20 / 50 were expected to be below the NOEL and no statistical significance was found in any test item concentration compared to control, no determination on EyL 10 / 20 / 50 was carried out.

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.

Any other information on results incl. tables

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		5523		32665		324419		1644249
		2		5523		35431		323728		2216333
		3		5523		34930		396677		2103279
		Mean		5523		34342		348275		1987954
50.0		1		5523		38308		330998		2066434
		2		5523		29662		316082		1847488
		3		5523		35809		435201		2208875
		Mean		5523		34593		360760		2040932
25.0		1		5523		37535		366302		2210801
		2		5523		32527		327304		1946417
		3		5523		36807		301117		1920339
		Mean		5523		35623		331574		2025852
12.5		1		5523		33251		270703		1617874
		2		5523		31216		326770		1831980
		3		5523		31996		337873		2080708
		Mean		5523		32154		311782		1843521
6.25		1		5523		32959		313465		1781652
		2		5523		24264		282142		1813212
		3		5523		21745		222982		1408066
		Mean		5523		26323		272863		1667643
Control		1		5523		26780		260756		1786195
		2		5523		33730		312892		1880926
		3		5523		33828		316201		1993535
		4		5523		30443		291536		1764908
		5		5523		37120		311983		1787381
		6		5523		28146		332579		2015464
		Mean		5523		31675		304325		1871402

 

 

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		1.90	2		1638726	12
	2		2.00	-3		2210810	-18
	3		1.98	-2		2097756	-12
	Mean	(-)	1.96	-1	(-)	1982431	-6
50.0	1		1.98	-2		2060911	-10
	2		1.94	0		1841965	1
	3		2.00	-3		2203352	-18
	Mean	(-)	1.97	-1	(-)	2035409	-9
25.0	1		2.00	-3		2205278	-18
	2		1.96	-1		1940894	-4
	3		1.95	0		1914816	-3
	Mean	(-)	1.97	-1	(-)	2020329	-8
12.5	1		1.89	2		1612351	14
	2		1.94	0		1826457	2
	3		1.98	-2		2075185	-11
	Mean	(-)	1.94	0	(-)	1837998	1
6.25	1		1.93	1		1776129	5
	2		1.93	1		1807689	3
	3		1.85	5		1402543	25
	Mean	(-)	1.90	2	(-)	1662120	11
Control	1		1.93			1780672
	2		1.94			1875403
	3		1.96			1988012
	4		1.92			1759385
	5		1.93			1781858
	6		1.97			2009941
	Mean		1.94			1865879

 

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	1.58	2.28	1.92	1.93	0.349	18.1	15.1
	2	1.81	2.23	1.79	1.94	0.246	12.7
	3	1.81	2.24	1.84	1.96	0.236	12.0
	4	1.71	2.26	1.80	1.92	0.296	15.4
	5	1.91	2.13	1.75	1.93	0.193	10.0
	6	1.63	2.47	1.80	1.97	0.444	22.6
				Mean	1.94
				SD ±	0.02
				CV [%]	1.01

 

SD = Standard deviation CV = Coefficient of variation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, Disperse Yellow 184.1 was found not to inhibit the growth of the freshwater green Effect values were determined as follows (based on nominal test item loadings):
The EL50-values for both inhibition of growth rate (ErL50) and yield (EyL50) after 72 hours were > 100% of the saturated solution. The NOEL-values for both inhibition of growth rate and yield after 72 hours were 100% of the saturated solution.

Executive summary:

The toxicity of Disperse Yellow 184.1 to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016 C.3 from 2018-04-16 to 2018-04-20, with the definitive exposure phase from 2018-04-17 to 2018-04-20 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 5523 cells/mL. A saturated solution with a nominal loading of 100 mg test item/L was prepared with dilution water 24 hours prior to the start of the exposure. An appropriate amount of the test item was weighed out. The saturated solution was stirred for 24 hours (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). The Tyndall effect was negative.

 

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 test item concentration and six replicates for the control.The test media were clear and colourless throughout the test period. The environmental conditions were within the acceptable limits.

The measured concentrations of the test item were all < 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 loadings of DisperseYellow 184.1.

 

NOEL, LOEL, EL_x-values ofDisperseYellow 184.1(0 - 72 hours)

                  based on the nominal test item loadings

	Growth Rate Inhibition nominal test item loadings [% saturated solution]
NOEL	100
LOEL	> 100
ErL10	> 100
ErL20	> 100
ErL50	> 100
	Yield Inhibition nominal test item loadings [% saturated solution]
NOEL	100
LOEL	> 100
EyL10	> 100
EyL20	> 100
EyL50	> 100