9(or 10)-methoxy-7H-benzimidazo[2,1-a]benz[de]isoquinolin-7-one

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 2018-07-09 to 2018-07-16, with the definitive exposure phase from 2018-07-10 to 2018-07-13

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:

9(or 10)-methoxy-7H-benzimidazo[2,1-a]benz[de]isoquinolin-7-one

EC Number:

269-535-7

EC Name:

9(or 10)-methoxy-7H-benzimidazo[2,1-a]benz[de]isoquinolin-7-one

Cas Number:

68296-59-3

Molecular formula:

C19H12N2O2

IUPAC Name:

9(or 10)-methoxy-7H-benzimidazo[2,1-a]benz[de]isoquinolin-7-one

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 71 was analytically verified via HPLC-DAD in the saturated solution, the dilutions and the control at the start of the exposure (0 hours) and at the end of the exposure (72 hours).
The samples were analysed with an HPLC-DAD method implemented under non-GLP but documented finally in the GLP raw data. The Method was validated according to Annex I.

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 ± 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 dilution water (see Table 2). The saturated solution was stirred for 24 ± 1 hour (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), 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. The saturated solution and four dilution levels out of the saturated solution were tested in a geometrical series with a dilution factor of √10: 1.00 - 3.16 - 10.0 - 31.6 - 100% of the saturated solution. The concentrations are based on the results of a non-GLP preliminary range finding test. For results see Annex II.

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): 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.: 24.0 mean value: 23.0

pH:

Nominal test item loading pH-values
[% of the saturated solution] Start; 0 hours End; 72 hours
100 7.31 7.53
31.6 7.40 8.05
10.0 7.48 8.60
3.16 7.60 8.99
1.00 7.76 9.15
Control 8.00 9.18

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 6442 cells/mL
- Control end cells density: Mean 1518947 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: 6031 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.:

31.6 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.:

80.1 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI: 75.3 - 85.2

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

45 other: % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: CI:32.0 - 97.8

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 2018-03-26 to 2018-03-29. The reference item toxicity was 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 1.09 0.759 ± 0.555
95% confidence interval 0.842 – 1.31
Yield inhibition
EyC50 0.548 0.401 ± 0.297
95% confidence interval 0.439 – 0.660
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 and third order polynomial 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) 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		6442		7189		12586		31379
		2		6442		8396		14041		30659
		3		6442		10466		21486		53165
		Mean		6442		8684		16038		38401
31.6		1		6442		21307		176240		1298360
		2		6442		21613		150799		1220459
		3		6442		21973		156231		1205117
		Mean		6442		21631		161090		1241312
10.0		1		6442		22874		160104		1513245
		2		6442		25329		170421		1245780
		3		6442		27585		197311		1639604
		Mean		6442		25263		175945		1466210
3.16		1		6442		29802		217817		1514486
		2		6442		17012		138834		1175973
		3		6442		25657		168326		1373580
		Mean		6442		24157		174992		1354680
1.00		1		6442		25736		199079		1722371
		2		6442		23368		179080		1467319
		3		6442		28747		181622		1399894
		Mean		6442		25950		186594		1529861
Control		1		6442		25068		196328		1499095
		2		6442		25748		160968		1364345
		3		6442		25887		198970		1407342
		4		6442		26153		169100		1568456
		5		6442		26297		219009		1434699
		6		6442		26548		208165		1839743
		Mean		6442		25950		192090		1518947

 

 

 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		0.528	71		24937	98
	2		0.520	71		24217	98
	3		0.704	61		46723	97
	Mean	(+)	0.584	68	(+)	31959	98
31.6	1		1.77	3		1291918	15
	2		1.75	4		1214017	20
	3		1.74	4		1198675	21
	Mean	(-)	1.75	4	(-)	1234870	18
10.0	1		1.82	0		1506803	0
	2		1.76	4		1239338	18
	3		1.85	-1		1633162	-8
	Mean	(-)	1.81	1	(-)	1459768	3
3.16	1		1.82	0		1508044	0
	2		1.74	5		1169531	23
	3		1.79	2		1367138	10
	Mean	(-)	1.78	2	(-)	1348238	11
1.00	1		1.86	-2		1715929	-13
	2		1.81	1		1460877	3
	3		1.79	1		1393452	8
	Mean	(-)	1.82	0	(-)	1523419	-1
Control	1		1.82			1492653
	2		1.79			1357903
	3		1.80			1400900
	4		1.83			1562014
	5		1.80			1428257
	6		1.89			1833301
	Mean		1.82			1512505

Negative inhibition = increase of growth

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	1.36	2.06	2.03	1.82	0.397	21.8	21.2
	2	1.39	1.83	2.14	1.79	0.378	21.2
	3	1.39	2.04	1.96	1.80	0.353	19.7
	4	1.40	1.87	2.23	1.83	0.414	22.6
	5	1.41	2.12	1.88	1.80	0.363	20.1
	6	1.42	2.06	2.18	1.89	0.410	21.8
				Mean	1.82
				SD ±	0.04
				CV [%]	1.98

 

SD = Standard deviation CV = Coefficient of variation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, Disperse Yellow 71 was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on nominal test item loadings): The EL50-values with 95% confidence intervals for inhibition of growth rate (ErL50) and yield (EyL50) after 72 hours were 80.1 (75.3 – 85.2) and 45.0 (32.0 – 97.8) % of the saturated solution, respectively. The NOEL-values for both inhibition of growth rate and yield after 72 hours were 31.6% of the saturated solution.
The substance does not cause any adverse effect to freshwater green algae up to the limit of water solubility and far beyond.

Executive summary:

The toxicity of Disperse Yellow 71 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 2018-07-09 to 2018-07-16, with the definitive exposure phase from 2018-07-10 to 2018-07-13, 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 6442 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 24hours (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 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 √10:1.00 - 3.16 - 10.0 - 31.6 - 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 throughout the test period.The environmental conditions were within the acceptable limits.

The measured concentrations of the test item in the saturated solution were between < LOD to 12.8 µg/L at the start of the exposure (0 hours) and < LOD to 8.66 µg/L at the end of the exposure period (72 hours).

 

All effect values given are based on the nominal test item loadings of Disperse Yellow 71.

 

NOEL, LOEL, EL_x-values and 95% Confidence Intervals of DisperseYellow 71 (0 - 72 hours)

                  based on the nominal test item loadings

	Growth Rate Inhibition nominal test item loadings [% saturated solution]
NOEL	31.6
LOEL	100
ErL10	35.9 (30.2 – 41.2)
ErL20	47.0 (41.9 – 51.9)
ErL50	80.1 (75.3 – 85.2)
	Yield Inhibition nominal test item loadings [% saturated solution]
NOEL	31.6
LOEL	100
EyL10	25.5 (< 1.00 – 96.1)
EyL20	32.5 (10.5 – 96.5)
EyL50	45.0 (32.0 – 97.8)