[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 fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2017-05-06 to 2017-05-12 with the definitive exposure period from 2017-05-08 to 2017-05-12 at the test facility.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

According to OECD and GLP

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 203 (Fish, Acute Toxicity Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

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:

Test item
C.I. Pigment Orange 68


Preparation of the saturated solution
The saturated solution (1.00 mg/L test item was weighed out) was prepared with dilution water 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. This procedure was repeated for every water renewal.

Test concentration
The saturated solution was tested in a limit test. The selection of the test concentration is based on the derivation of a threshold concentration (TC) from the results of an algae toxicity test and a Daphnia acute toxicity test, both EC50 > saturated solution prepared with a nominal loading of 1.00 mg test item/L
(algae: SCHEERBAUM, 2015; daphnia: NOACK, 2015).


Control
7 fish in dilution water (without test item) were tested under the same test conditions as the test replicates.

Test organisms

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: All fish used in the test were gained at NOACK LABORATORIEN from a single brood stock. (supplier: Umweltbundesamt, Schichauweg 58, D-12307 Berlin)
- Length at study end (length definition, mean, range and SD): Average body length: 2.37 cm
- Weight at study end (mean and range, SD): Average body weight: 0.12 g
- Method of breeding:Holding was performed at the test facility at 23 +/- 2 °C and diffuse light (0.1 - 10 µmol photons • m-2 • s-1, natural photoperiod). The water was changed at least once per week. The dissolved oxygen concentration was more than 80 % of the air saturation value. No disease treatments were administered throughout holding and testing. Food was provided 3 times per week. The amount of food was 4 % of the fish body weight per feeding day. The test fish were not fed 24 h before the test started.
- Feeding during test: No feeding during test


ACCLIMATION
- Acclimation period: Zebrafish with at least 12 days of acclimatisation and mortality < 5 % within these days before the study starts were used in the test.
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: 4 % of the fish body weight per feeding day. The test fish were not fed 24 h before the test started. Food: Sera Vipan; SERA GMBH, D-52518 Heinsberg
- Feeding frequency: 3 times per week.
- Health during acclimation (any mortality observed): No mortality observed, no disease treatments were administered throughout holding and testing.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Test conditions

Hardness:

Total Hardness at day 0: 59 mg CaCO3/L

Test temperature:

Please refer to "Any other information on materials and methods"

pH:

Please refer to "Any other information on materials and methods"

Dissolved oxygen:

Please refer to "Any other information on materials and methods"

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods incl. tables"

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria
- Type (delete if not applicable):
- Material, size, headspace, fill volume: Glass aquaria of 3 L were used, (dimensions: 11.5/13/20 cm), 2.5 L test volume
- Aeration: Gentle aeration was provided
- Renewal rate of test solution (frequency/flow rate): A semi-static test with one water renewal of the test media after 48 hours was performed.
- Control: 7 fish in dilution water (without test item) were tested under the same test conditions as the test replicates
- Reference item: No reference item is recommended for this test according to the guideline.
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control and pH-control (replicates): 1
- Biomass loading rate: 0.336 g fish per L test solution


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water of local origin was used for holding and testing. The water was filtered on activated charcoal and aerated for at least 24 h to remove possible chlorine.
Nominal water parameters:
Total hardness: 10 - 250 mg CaCO3/L
pH-value: 6.0 - 8.5
Acidity: 0.1 mmol/L
(recent measurement 2017-04-26)
Acid capacity: 0.6 mmol/L
(recent measurement 2017-04-26)
Conductivity: 151 µS/cm
(recent measurement 2017-04-26)



- Culture medium different from test medium: no
- Intervals of water quality measurement:The pH-value, temperature and oxygen saturation was measured in all test vessels at test start and end and every
24 hours and from fresh and old test media after renewal. Total hardness was determined at the start of the exposure out of the control. During the test the water temperature was recorded continuously (once per hour) with a data logger. The light intensity on the surface of the test aquaria was measured at the start of the exposure.



OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: A photoperiod of 16 h light / 8 h dark corresponding to natural daylight hours occurred during the course of the study.
- Light intensity: 7 - 750 Lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Fish were considered dead if there was no visible movement (e.g. gill covers movement) and if touching of the caudal peduncle produced no reaction. Records were kept of visible abnormalities (e.g. loss of equilibrium, swimming behaviour, respiratory function, pigmentation, etc.). Observations were made after 2, 24, 48, 72 and 96 h.


TEST CONCENTRATIONS
- Range finding study: None

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 2

Key result

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: Not observed
- Mortality of control: 0%
- Other adverse effects control: Not observed
- Abnormal responses: Not observed
- Any observations (e.g. precipitation):

Results with reference substance (positive control):

No reference substance tested

Reported statistics and error estimates:

Evaluation
The LC0 and LC100 after 96 h was derived from the raw data of the limit concentration. LC50-values do not have to be calculated in a limit test.


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 variations may occur from these figures. The following software was used for calculations:
Excel, MICROSOFT CORPORATION

Any other information on results incl. tables

Sublethal observations / clinical signs:

Biological Data

 

All effect levels are given based on the nominal threshold concentration of the test item after 96 hours.The LC₀after 96 hours was ≥ 1.00 mg/L..

 

Observations of Sub Lethal Effects and Normal Behaviour in the Test Vessels

Saturated solution with a nominal loading of [mg/L]	Effect *	Number of fish effected at observation time [hours]
		2	24	48	72	96
1.00	(1)	7	7	7	7	7
Control	(1)	7	7	7	7	7

              *) The numbers in brackets correspond to the following observations: 

                         (1)      = Normal behaviour

 

 

Cumulative Mortality [%] in the Test Vessels

Saturated solution with a nominal loading of [mg/L]	Cumulative Mortality [%] after different exposure periods [hours]
	2	24	48	72	96
1.00	0	0	0	0	0
Control	0	0	0	0	0

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

All effect levels are given based on the saturated solution with a nominal loading of 1 mg test item/L. The saturated solution of the test item found to have no effect on the vitality of zebrafish. In conclusion, the LC0 corresponds to the saturated solution with a nominal loading 1.00 mg test item/L.

Executive summary:

The acute toxicity of the test item (batch no.DEB2188394) to fish (zebrafish) was determined according to the principles of the OECD-Guideline for Testing of Chemicals No. 203 (1992) and EU Council Regulation (EC) No. 440/2008/ Method C.1 (2008) from 2017-05-06 to 2017-05-12 with the definitive exposure period from 2017-05-08 to 2017-05-12 at the test facility.

 

An acute toxicity test with the saturated solution (nominal loading of 1.00 mg/L) was conducted under semi-static conditions with one water renewal of the test media after 48 hours of exposure. Duration of the test was 96 hours. Seven test organisms were exposed to the threshold concentration and the control. Water quality parameters (temperature, pH-value and oxygen-saturation) measured at 0, 24, 48, 72 and 96 hours were determined to be within the acceptable limits.

 No analytical monitoring will be carried out, since no suitable method for the determination of thetest item could be established. No suitable column was found which is compatible for liquid chromatography.

All validity criteria of the test guideline were met.

 

LC-Values (2 – 96 hours) of the Test Item

                   Based on the saturated solution with a nominal loading of 1 mg/L

LC100= Lowest test item concentration with 100 % mortality after 96 h	> 1.00 mg/L
LC0= Highest test item concentration with 0 % mortality after 96 h	1.00 mg/L