[1,3-dihydro-5,6-bis[[(2-hydroxy-1-naphthyl)methylene]amino]-2H-benzimidazol-2-onato(2-)-N5,N6,O5,O6]nickel

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:

2015-09-23 to 2015-09-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to OECD 202 and GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

[1,3-dihydro-5,6-bis[[(2-hydroxy-1-naphthyl)methylene]amino]-2H-benzimidazol-2-onato(2-)-N5,N6,O5,O6]nickel

EC Number:

255-965-2

EC Name:

[1,3-dihydro-5,6-bis[[(2-hydroxy-1-naphthyl)methylene]amino]-2H-benzimidazol-2-onato(2-)-N5,N6,O5,O6]nickel

Cas Number:

42844-93-9

Molecular formula:

C29H18N4NiO3

IUPAC Name:

Nickel, [1,3-dihydro-5,6-bis[[[2-(hydroxy-kO)-1-naphthalenyl]methylene]amino-kN]-2H-benzimidazol-2-onato(2-)]-, (SP-4-2)-

Test material form:

solid: nanoform

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

Preparation of the saturated solution
The saturated solution (1.00 mg/L test item was weighed out) was
prepared with dilution water (see below) two days prior to the start of the exposure (0 hours).

Treatment
The test item was dispersed in the dilution water by stirring with approximately 1100 rpm for 48 hours at room temperature. After completion of stirring, the
test item dispersion was filtered with a membrane filter (0.45 µm, RC, MACHEREY-NAGEL) to achieve the saturated solution. Centrifugation was found to be
unsuitable.

Test concentration
The effects of the saturated solution of the test item were tested in a limit test.

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

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system
Daphnia magna STRAUS (Clone 5)

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

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

Breeder
DR.U.NOACK-LABORATORIEN, 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. 20 µEm-2  s-1

Culture medium
Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L, is used. The composition of the culture medium is presented
in Table 2.

Culture feeding
The culture daphnids are 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 are cultured at the test facility.

Origin of the food algae
Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, 37073 Göttingen, Germany

 

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

2015-09-23 (0h) 274 mg/L CaCO3

Test temperature:

18 - 22°C, constant within ± 1°C
2015-09-23 20.4°C

pH:

Water Quality Parameters at the Start of the Exposure (0 hours)
(measured in one additional replicate (without daphnids) of the saturated solution and the control)

PV-Echtorange 6RL
Nominal loading
of the test item
[mg/L] pH-value Dissolved O2 concentration[ mg/L]

1.00
(Saturated solution) 7.98 6.89
Control 7.99 8.62


Water Quality Parameters at the End of the Exposure (48 hours)
(measured in all replicates (containing daphnids) of the saturated solution and the control)

PV-Echtorange 6RL
Nominal loading
of the test item
[mg/L] pH-values Dissolved O2 concentration [mg/L]

Replicates Replicates
1 2 3 4 1 2 3 4

1.00
(Saturated solution) 7.82 7.83 7.84 7.85 8.41 8.47 8.56 8.58
Control 7.73 7.78 7.81 7.83 8.64 8.68 8.71 8.70

Dissolved oxygen:

Water Quality Parameters at the start of the Exposure (0 hours)
(measured in one additional replicate per loading level and control)

Water Quality Parameters at the Start of the Exposure (0 hours)
(measured in one additional replicate (without daphnids) of the saturated solution and the control)

PV-Echtorange 6RL
Nominal loading
of the test item
[mg/L] pH-value Dissolved O2 concentration[ mg/L]

1.00
(Saturated solution) 7.98 6.89
Control 7.99 8.62


Water Quality Parameters at the End of the Exposure (48 hours)
(measured in all replicates (containing daphnids) of the saturated solution and the control)

PV-Echtorange 6RL
Nominal loading
of the test item
[mg/L] pH-values Dissolved O2 concentration [mg/L]

Replicates Replicates
1 2 3 4 1 2 3 4

1.00
(Saturated solution) 7.82 7.83 7.84 7.85 8.41 8.47 8.56 8.58
Control 7.73 7.78 7.81 7.83 8.64 8.68 8.71 8.70

Details on test conditions:

Test method
Static procedure

Test duration
48 hours

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

Test volume
20 mL

Dilution water ISO Test water, according to OECD 202, Annex 3
Component Concentration [mg/L]
KCl 5.75
NaHCO3 64.8
CaCl2  2 H2O 294
MgSO4  7 H2O 123

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

Age of the daphnidsat the start of the exposure
2 to 24 hours old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels latest 22 hours before the start of the exposure and discarded. The juveniles born within this period of max. 22 hours preceding the exposure were used for the test after an
acclimatisoation phase of at least 2 hours in the dilution water. No first brood progeny was used for the test

Acclimatisation
At least 2 hours in dilution water
 
Application
Per replicate, 20 g test solution were weighed out into the 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. 20 µEm-2  s-1 (max. 1340 lx)

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

Feeding
The daphnids were not fed during the study.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EL10

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EL10

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Effect concentrations 3

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Effect concentrations 4

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Effect concentrations 5

Duration:

48 h

Dose descriptor:

other: NOEL

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Effect concentrations 6

Duration:

48 h

Dose descriptor:

other: LOEL

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Effect concentrations 7

Duration:

24 h

Dose descriptor:

EL100

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Effect concentrations 8

Duration:

48 h

Dose descriptor:

EL100

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Details on results:

See below

Results with reference substance (positive control):

A reference test was conducted as an acute immobilisation test (acc. to AQS P 9/2 and OECD 202) under static conditions with a test duration of 24 hours once per month in order to prove the validity of the test system and test conditions at the test facility. The results of the most recent test are presented in section 8.2.

Reference item Potassium dichromate p.a. (SIGMA)

Purity 100.0 %

Batch No. MKBQ9179V

Storage stability 2015-12-29

Test concentrations 1.00 – 2.00 – 4.00 mg/L

Ranges of validity 0.6 mg/L < EC50 (24 h) < 2.4 mg/L, according to AQS P 9/2 (clone 5),
0.6 mg/L < EC50 (24 h) < 2.1 mg/L, according to OECD 202 (clone A)

Test duration 2015-09-02 to 2015-09-03

Reported statistics and error estimates:

Methods of evaluation The effects of the saturated solution were deduced directly from the observed immobilisation rates. According to OECD Guidance Document No 23, if an effect cannot be detected in a test with a saturated solution, the result have to be reported as no toxic effects at saturation.
Therefore, the effect levels (NOEL, LOEL, EL10 / 50 / 100) were given based on the nominal loading of the test item.

EC-values and statistical An EC50-value was calculated only for the reference item by
analyses for reference item sigmoidal dose-response regression. The respective 95 % confidence limits for the EC50 were calculated from the standard error and the t-distribution. All calculations were carried out from the best-fit values with the software GraphPad Prism5.
An EC50 for the test item could not be calculated, since a limit test was carried out with a saturated solution of the test item resulting in 0 % immobilisation.

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 Prism5, GRAPHPAD SOFTWARE, INC.
- Excel, MICROSOFT CORPORATION

Any other information on results incl. tables

Composition of the Culture Medium

                     according toElendt(1990)

Component	Concentration [mg/L]
CaCl2x 2 H2O	147
MgSO4x 7 H2O	123
KCl	5.80
NaHCO3	64.8
Na2SiO3	4.30
NaNO3	0.27
KH2PO4	0.14
K2HPO4	0.18
Na2EDTA x 2 H2O	5.00
FeSO4x 7 H2O	1.99
H3BO3	0.29
MnCl2x 4 H2O	0.36
LiCl	0.30
SrCl2x 6 H2O	0.15
RbCl	0.071
NaBr	0.016
Na2MoO4x 2 H2O	0.063
CuCl x 2 H2O	0.017
ZnCl2	0.013
CoCl2x 6 H2O	0.010
KJ	0.00325
Na2SeO3	0.00219
NH4VO3	0.000575
Thiaminhydrochloride	0.075
Cyanocobalamin	0.0010
Biotin	0.00075
pH	8.2±0.8

 

ISO Test water, according to OECD 202, Annex 3

Component	Concentration [mg/L]
KCl	5.75
NaHCO3	64.8
CaCl2×2 H2O	294
MgSO4×7 H2O	123

Immobilisation Rates of the definitive Testafter 24 and 48 hours of Exposure

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

PV-Echtorange 6RL Nominal loading of the test item   [mg/L]	IMMOBILISATION [%]
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	MV	1	2	3	4	MV
1.00 (Saturated solution)	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

Water Quality Parameters at the Start of the Exposure (0 hours)

             (measured in one additional replicate (without daphnids) of the saturated solution and the control)

PV-Echtorange 6RL Nominal loading of the test item [mg/L]	pH-value	Dissolved O2concentration   [mg/L]
1.00 (Saturated solution)	7.98	6.89
Control	7.99	8.62

Water Quality Parameters at the End of the Exposure (48 hours)

             (measured in all replicates (containing daphnids) of the saturated solution and the control)

PV-Echtorange 6RL Nominal loading of the test item [mg/L]	pH-values				Dissolved O2concentration [mg/L]
	Replicates				Replicates
	1	2	3	4	1	2	3	4
1.00 (Saturated solution)	7.82	7.83	7.84	7.85	8.41	8.47	8.56	8.58
Control	7.73	7.78	7.81	7.83	8.64	8.68	8.71	8.70

 

 

   Water Quality Parameters of the Dilution Waterprior to the Start of the Exposure (0 hours)

Dilution water dated:	pH-value	Dissolved O2concentration [mg/L]	Temperature   [°C]	Conductivity   [µS/cm]	Total hardness   [mg CaCO3/L]
2015-09-23	7.99	8.62	20.4	645	274

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In the saturated solution with a nominal loading of 1.00 mg/L of the test item no effects were observed on Daphnia magna.

Executive summary:

     Summary

 

In the acute immobilisation test with Daphnia magna (STRAUS), the effects of the saturated solution with a nominal loading of 1.00 mg/L of the test item were determined according to OECD 202 (2004).The limit test was conducted under static conditions over a period of 48 hours.

The test item is sparingly soluble in water (< 0.02 mg/L at 20 °C). Therefore, a saturated solution (1.00 mg/L) was prepared with dilution water (ISO test water, acc. to OECD 202, Annex 3) by stirring with approximately 1100 rpm for 48 hours at room temperature followed by filtration with a membrane filter (0.45 µm, RC,Macherey-Nagel) to remove undissolved test item and to achieve the saturated solution.Centrifugation was found to be unsuitable for removing the undissolved test item completely. The saturated solution was visually clear throughout the exposure period of 48 hours.

Twenty daphnids were exposed to the saturated solutionand the control.

No analytical monitoring was performed during the test period in agreement with the sponsor, since no suitable method for the determination of the test item could be established. No suitable column was found which is compatible for liquid chromatography.Exposures are expressed in terms of the original concentration of the test item in water at the start of the mixing period (loading rate). The EL-values are presented.

Water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 hours) and at the end of the exposure (48 hours), were within the acceptable limits. The validity criteria of the test guideline were fulfilled.

 

 

 

In the saturated solution with a nominal loading of1.00 mg/Lof the test item

no effects were observed onDaphnia magna.

 

 

 

Table1:   EL₁₀-, EL₅₀-EL₁₀₀-Values, NOEL and LOEL

                  (based on the nominal loading levels of the test item)

Endpoints	Test duration [hours]
		Nominal loading level of the test item [mg/L]
EL10 / 50 / 100	24	> 1.00
	48	> 1.00
NOEL	48	1.00
LOEL	48	> 1.00