2-(3-methoxypropyl)-9(or 10)-methylbenzimidazo[2,1-b]benzo[lmn][3,8]phenanthroline-1,3,6(2H)-trione

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 2017-03-27 to 2017-03-31, with the definitive exposure phase from 2017-03-28 to 2017-03-31 at the test facility

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

Test material

Test material information

Constituent 1

Reference substance name:

2-(3-methoxypropyl)-9(or 10)-methylbenzimidazo[2,1-b]benzo[lmn][3,8]phenanthroline-1,3,6(2H)-trione

EC Number:

258-094-6

EC Name:

2-(3-methoxypropyl)-9(or 10)-methylbenzimidazo[2,1-b]benzo[lmn][3,8]phenanthroline-1,3,6(2H)-trione

Cas Number:

52673-15-1

Molecular formula:

C25H19N3O4

IUPAC Name:

17-(3-methoxypropyl)-6(or7)-methyl-3,10,17-triazahexacyclo[13.6.2.0²,¹.0,.0¹²,²².0¹,²³]tricosa-1(21),2,4(9),5,7,12(22),13,15(23),19-nonaene-11,16,18-trione

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 Orange 32 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 levels.
The measured test item concentration was below the LCL in the saturated solution, the dilutions and the control.
At the end of the exposure (72 hours), no analytical monitoring was carried out, because a measured test item concentration below LCL was observed at the start of the exposure.

Test solutions

Vehicle:

no

Details on test solutions:

Test item
Disperse Orange 32

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. 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 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.

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 7.92 8.03
50.0 7.89 8.24
25.0 7.90 8.70
12.5 7.93 9.16
6.25 7.96 9.28
Control 8.12 9.24

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 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 1416910 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: 5547 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)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

other: NOEL

Effect conc.:

12.5 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.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

27.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI: 20.1 - 34.8

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

65.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI: 58.5 - 76.1

Effect concentrations 5

Duration:

72 h

Dose descriptor:

other: NOEL

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 6

Duration:

72 h

Dose descriptor:

other: LOEL

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 7

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

13.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: CI: 8.31 - 17.2

Effect concentrations 8

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

30.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: CI: 26.9 - 35.2

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 orange throughout the exposure period. The Tyndall effect negative at the saturated solution.
- 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 2016-10-11 to 2016-10-14. 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 0.763 ± 0.514
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 statistical analyses
The NOEL and LOEL were determined by calculation of statistically significant differences of growth rates and yield.
The following statistical tests were conducted:

Shapiro-Wilk’s test on normal distribution was done with a significance level 0.01.
Levene’s test on variance homogeneity was done with a significance level 0.01.
Multiple sequentially-rejective Welsh-t-test after Bonferroni-holm was done with a significance level 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.
•ToxRat Version 3.2.1, ToxRat Solutions GmbH

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		6839		2819		LOQcell density		4615
		2		6839		3021		2836		8337
		3		6839		LOQcell density		3097		11600
		Mean		6839		n.a.		n.a.		8184
50.0		1		6839		8486		32992		230497
		2		6839		9835		31321		231246
		3		6839		10324		53912		383304
		Mean		6839		9548		39408		281682
25.0		1		6839		19084		151234		962286
		2		6839		22458		153616		966090
		3		6839		14970		129114		825642
		Mean		6839		18837		144655		918006
12.5		1		6839		26508		245033		1371490
		2		6839		26692		230181		1346210
		3		6839		19866		162192		1045124
		Mean		6839		24355		212469		1254275
6.25		1		6839		28199		262147		1515382
		2		6839		22786		223149		1307461
		3		6839		24846		275923		1467630
		Mean		6839		25277		253740		1430158
Control		1		6839		24954		228823		1348568
		2		6839		26964		264895		1423649
		3		6839		22379		220583		1408024
		4		6839		34671		297947		1483808
		5		6839		28693		271894		1440561
		6		6839		26683		255906		1396847
		Mean		6839		27391		256675		1416910

LOQ_{cell density}= Limit of quantification of the cell density (2736 cells/mL).             n.a.       = not applicable

 

 

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		-0.131	107		-2224	100
	2		0.066	96		1498	100
	3		0.176	90		4761	100
	Mean	(+)	0.037	98	(+)	1345	100
50.0	1		1.17	34		223658	84
	2		1.17	34		224407	84
	3		1.34	25		376465	73
	Mean	(+)	1.23	31	(+)	274843	81
25.0	1		1.65	7		955447	32
	2		1.65	7		959251	32
	3		1.60	10		818803	42
	Mean	(+)	1.63	8	(+)	911167	35
12.5	1		1.77	1		1364651	3
	2		1.76	1		1339371	5
	3		1.68	6		1038285	26
	Mean	(-)	1.74	2	(-)	1247436	12
6.25	1		1.80	-1		1508543	-7
	2		1.75	2		1300622	8
	3		1.79	-1		1460791	-4
	Mean	(-)	1.78	0	(-)	1423319	-1
Control	1		1.76			1341729
	2		1.78			1416810
	3		1.78			1401185
	4		1.79			1476969
	5		1.78			1433722
	6		1.77			1390008
	Mean		1.78			1410071

 

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	1.29	2.22	1.77	1.76	0.461	26.2	25.0
	2	1.37	2.29	1.68	1.78	0.464	26.1
	3	1.19	2.29	1.85	1.78	0.555	31.3
	4	1.62	2.15	1.61	1.79	0.310	17.3
	5	1.43	2.25	1.67	1.78	0.420	23.5
	6	1.36	2.26	1.70	1.77	0.454	25.6
				Mean	1.78
				SD ±	0.01
				CV [%]	0.60

 

SD = Standard deviation CV = Coefficient of variation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, Disperse Orange 32 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 both inhibition of growth rate (ErL50) and yield (EyL50) after 72 hours were 65.6 (58.5 – 76.1)% and 30.7 (26.9 – 35.2)% of the saturated solution, respectively. The NOEL-values for both inhibition of growth rate and yield after 72 hours were 12.5% of the saturated solution, respectively.

Executive summary:

The toxicity of Disperse Orange 32 to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009 C.3 from 2017-03-27 to2017-03-31, with the definitive exposure phase from 2017-03-28 to 2017-03-31at 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 100 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. 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 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 throughout the test period with a concentration dependent orange color.The environmental conditionswere within the acceptable limits.

 

All effect values given are based on the nominal test item loadings ofDisperseOrange 32.

 

 NOEL, LOEL, EL_x-values and 95 % Confidence Intervals ofDisperseOrange 32(0 - 72 hours)

                  based on the nominal test item loadings

	Growth Rate Inhibition nominal test item loadings [% saturated solution]
NOEL	12.5
LOEL	25.0
ErL10	27.6 (20.1 – 34.8)
ErL20	39.6 (33.0 – 44.6)
ErL50	65.6 (58.5 – 76.1)
	Yield Inhibition nominal test item loadings [% saturated solution]
NOEL	12.5
LOEL	25.0
EyL10	13.1 (8.31 – 17.2)
EyL20	17.9 (14.4 – 21.6)
EyL50	30.7 (26.9 – 35.2)