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

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2018-07-02 to 2018-07-05, with the definitive exposure phase from 2018-07-03 to 2018-07-05

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

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 saturated solution and the control were analytically verified via HPLC-DAD in the fresh media at the start of the exposure (0 hours) and in the old media at the end of the exposure (48 hours).The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000).

Sampling for the analytical monitoring
At the start of the exposure (0 hours), samples were taken after preparation of the saturated solution and analyzed.
At the end of the exposure (48 hours), samples for the analyses of the old media were taken directly from test replicates containing daphnids.

Criteria for the analytical monitoring (target)
Recoveries of the test item should be within ± 20% of the nominal or initially measured concentrations.

Test solutions

Vehicle:

no

Details on test solutions:

Limit loading level
A saturated solution with a nominal loading of 100 mg/L of the test item was tested in a limit test. The limit loading level was selected based on the results of a non-GLP preliminary range finding test.

Preparation of the saturated solution
A saturated solution (100 mg/L test item were weighed out) was prepared with dilution water one day before the start of the exposure (at day -1).
The test item was mixed with the dilution water by constant stirring with a magnetic stirrer at approximately 1100 rpm for 24 hours at room temperature. After completion of stirring, 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. The following filtrate, i.e. the saturated solution, was tested in a limit test. During filtration, the filter was always kept covered.
The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative.

Control
Dilution water without test item incubated under the same conditions as the test groups

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system:
Daphnia magna STRAUS, obtained from continuous laboratory cultures.

Reason for the selection of the test system
Daphnia magna is the preferred species in accordance with the test guideline.

Origin
Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany

Breeder
Noack Laboratorien GmbH, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany

Culture
In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20  2 °C, in an incubator, 16 hours illumination;
light intensity of max. 1500 lx

Culture medium
Elendt M4, according to OECD 202, Annex 3 (2004), is used. The composition of the culture medium is presented in the table below.

 

Composition of the Culture Medium Elendt M4
according to OECD 202, Annex 3 (2004)

Component Concentration [mg/L]
CaCl2 x 2 H2O 294
MgSO4 x 7 H2O 123
KCl 5.80
NaHCO3 64.8
Na2SiO3 x 5 H2O 7.47
NaNO3 0.274
KH2PO4 0.143
K2HPO4 0.184
Na2EDTA x 2 H2O 2.50
FeSO4 x 7 H2O 0.996
H3BO3 2.86
MnCl2 x 4 H2O 0.361
LiCl 0.306
SrCl2 x 6 H2O 0.152
RbCl 0.0710
NaBr 0.0160
Na2MoO4 x 2 H2O 0.0615
CuCl x 2 H2O 0.0168
ZnCl2 0.0130
CoCl2 x 6 H2O 0.0100
KI 0.00325
Na2SeO3 0.00219
NH4VO3 0.000575
Thiaminhydrochloride 0.075
Cyanocobalamin 0.0010
Biotin 0.00075
pH 8.2 to 0.8

Feeding of the culture stocks
The daphnids were fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 106 cells/mL. The algae were cultured at the test facility.

Study design

Test type:

static

Water media type:

freshwater

Remarks:

Elendt M4, according to OECD 202, Annex 3 (2004)

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

see any other information on materials and methods incl. tables

Test temperature:

see any other information on materials and methods incl. tables

pH:

see any other information on materials and methods incl. tables

Dissolved oxygen:

see any other information on materials and methods incl. tables

Salinity:

Not applicable, freshwater

Conductivity:

see any other information on materials and methods incl. tables

Nominal and measured concentrations:

Nominal test item concentration: 100 mg/L
Geometric mean measured test item concentrations: 15.2 µg/L

Details on test conditions:

Test method
The study was performed under static conditions.

Test duration
48 hours

Test vessels
Glass beakers (4 (ID) x 7 (H) cm), 50 mL capacity, loosely covered with watch glasses

Test volume
20 mL

Dilution water
Same composition as the culture medium

Number of daphnids and replicates
20 daphnids, divided into 4 replicates, each with 5 daphnids, were used for the the limit concentration and the control.

Age of the daphnids at the start of the exposure
Less than 24 hours old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.

Acclimatization
Acclimatization of the daphnids was not necessary, because the composition of the dilution water was equivalent to the culture medium.

Application
20 g test solution per replicate was weighed into each test vessel. This corresponds to 20 mL per test vessel. The daphnids were inserted with a small amount of dilution water by a pipette.

Test temperature (target)
18 - 22 °C, constant within ± 1 °C

Illumination (target)
Diffuse light, light intensity of max. 1500 lx

Photoperiod (target)
16/8 hours light/dark cycle

Feeding
The daphnids were not fed during the study.

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

other: EC10/50/100

Effect conc.:

> 15.2 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Duration 24 and 48 hours

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

other: EL10/50/100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Duration 24 and 48 hours

Results with reference substance (positive control):

The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) was determined after 24 hours from 2018 06-20 to 2018-06-21. For results of the most recent of the monthly performed reference tests, see Table 6.

EC50-Value (with 95% Confidence Limits) of the Reference Item Potassium dichromate
based on nominal concentrations mg/L, (0 - 24 hours)
Current Study Valid Range
EC50 2.03 mg/L 0.6 - 2.4 mg/L, acc. to AQS P 9/2 (02/2000); clone 5
95% confidence limits 1.00 - 4.00 mg/L 0.6 - 2.1 mg/L, acc. to OECD 202 (2004); clone A

Reported statistics and error estimates:

Methods of evaluation
The EC10 / 50 / 100-values and the EL10 / 50 / 100-values after 24 and 48 hours were empirically derived from the observation data. The effect levels were based on the nominal loading and the geometric mean measured concentration of the test item Disperse Yellow 71.

EC-values and statistical analyses for reference item
An EC50-value was calculated for the reference item by sigmoidal dose-response regression from the best-fit values with the software GraphPad Prism. The respective 95 % confidence limits for the EC50 were empirically derived from the observation data as follows: The highest concentration level without any effect (EC0) and the lowest concentration level causing 100% immobilization (EC100) were used as 95% confidence limits, since calculation was not possible by the software.

Software
All data were computer-processed and rounded for presentation. Consequently, minor variations may occur from the original figures if manual calculations based on the original figures are made subsequently. Calculations were made using the following software:
- GraphPad Prism, GRAPHPAD SOFTWARE, INC.
- Excel, MICROSOFT CORPORATION

Any other information on results incl. tables

  Biological Data

 

Immobilization Ratesafter 24 and 48 hours of Exposure in the Definitive Test

             (n = 20, divided into 4 replicates with 5 daphnids each)

Nominal loading of the test item in the saturated solution [mg/L]	Geometric mean measured test item concentration [µg/L]	IMMOBILIZATION [%]
		24 hours					48 hours
		Replicates					Replicates
		1	2	3	4	MV	1	2	3	4	MV
100	15.2	0	0	0	0	0	0	0	0	0	0
Control		0	0	0	0	0	0	0	0	0	0

 

Absolute Numbers of immobiledaphnids after 24 and 48 hours of Exposure in the Definitive Test

(n = 20, divided into 4 replicates with 5 daphnids each)

Nominal loading of the test item in the saturated solution [mg/L]	Geometric mean measured test item concentration [µg/L]	Number of immobile Daphnids / Total number of Daphnids
		24 hours					48 hours
		Replicates					Replicates
		1	2	3	4	Sum	1	2	3	4	Sum
100	15.2	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20
Control		0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20

 

 

 Additional Observations during the Definitive Test

 

The saturated solution was colourless andvisually clear throughout theexposure period.The Tyndall effect was negative.

 

 Water Quality Parameters

 

The measured water quality parameters(i.e. pH-value, dissolved oxygen concentration, total water hardness and water temperature) were within the acceptable limits. During the test period, the temperature in the incubator was 19 - 20 °C.

 

Measured Exposure Concentrations during the Definitive Test

 

The concentrations of the test item Disperse Yellow 71 were analytically verified via HPLC-DAD in the fresh media at the start of the exposure (0 hours) and in the old media at the end of the exposure (48 hours) in the saturated solution and in the control.

The measured concentrations of the test item in the saturated solution was 17.8 µg/L at the start of the exposure (0 hours) and 13.0 µg/L at the end of the exposure period (48 hours). The geometric mean measured concentration is 15.2 µg/L.

 

 Measured Concentrations and Percent of Initially Measured Concentration of the Test ItemDisperse Yellow71during the Definitive Test

Sampling date	Start of the exposure (0 hours)	End of the Exposure (48 hours)		Geometric mean measured test itemconcentration [µg/L]
Nominal loading of the test item in the saturated solution [mg/L]	Disperse Yellow71
	Meas. conc. [µg/L]	Meas. conc. [µg/L]	%
100	17.8	13.0	73	15.2
Control	< LOQ	< LOQ

Meas. conc.    = measured concentration of the test item, mean value of two injections

%                     = percentage of the initially measured concentration of the test item

LOQ                = limit of quantification (10 µg test item/L)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The saturated solution of the test item Disperse Yellow 71 in Elendt M4 medium with a nominal loading of 100 mg/L, which corresponds to a geometric mean measured concentration of 15.2 µg/L, caused no effects on Daphnia magna.
Based on the geometric mean measured concentrations of the test item Disperse Yellow 71, the 48 hours EC50 for Daphnia magna was > 15.2 µg/L. Based on the nominal loading of Disperse Yellow 71, the 48-hours EL50 for Daphnia magna was > 100 mg/L.

The substance does not cause any adverse effect to Daphnia magna up to the limit of water solubility and far beyond.

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item Disperse Yellow 71 were determined at the test facility according to OECD 202 (2004) from 2018-07-02 to 2018-07-05, with the definitive exposure phase from 2018-07-03 to 2018-07-05.

The study was conducted under static conditions over a period of 48 hours with a saturated solution with a nominal loading of100 mg/Lof the test item Disperse Yellow 71. The saturated solution was colourless andvisually clear throughout the exposure period.The Tyndall effect was negative.

Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to the saturated solutionand the control.

The concentrations of the test item Disperse Yellow 71 were analytically verified via HPLC-DAD in the fresh media at the start of the exposure (0 hours) and in the old media at the end of the exposure (48 hours) in the saturated solution and in the control.

The measured concentrations of the test item in the saturated solution was 17.8 µg/L at the start of the exposure (0 hours) and 13.0 µg/L at the end of the exposure period (48 hours). The geometric mean measured concentration is 15.2 µg/L.

The measured test item concentration at the end of the exposure was not within ± 20% of the initial concentration of the test item over the test period. The effect levels given are based on the nominal loading and the geometric mean measured concentration of the test item DisperseYellow 71.

The saturated solution of the test item Disperse Yellow 71 in Elendt M4 medium with a nominal loading of 100 mg/L, which corresponds to a geometric mean measured concentration of 15.2 µg/L, caused no effects on Daphnia magna.

The validity criteria of the test guideline were fulfilled.

 

 EC_{10 / 50 /100}-Values and EL_{10 / 50 /100}-Values of the Test Item DisperseYellow 71

(based on the geometric mean measured concentration and the nominal loading of the test item)

Effect levels		Test duration   [hours]		DisperseYellow 71
			Geometric mean measured test item concentration [µg/L]		Nominal loading of the test item [mg/L]
EC10 / 50 / 100		24		> 15.2		─
		48		> 15.2		─
EL10 / 50 / 100		24		─		> 100
		48		─		> 100