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

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:

2015-08-30 to 2015-09-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to OECD 201 and GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition 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

Details on sampling:

No analytical monitoring was carried out since no suitable method for the determination of the test item could be established.

Test solutions

Vehicle:

no

Details on test solutions:

Preparation of the saturated solution:
The saturated solution (1.00 mg/L test item were weighed out) was prepared with demineralized water two days prior to the start of the exposure (0 h). The dispersion was stirred for 48 hours (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 filtrate were discarded. The filtration was interrupted for ca. 15 minutes to allow for adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. After the separation treatment the components of the dilution water were added to the saturated solution. The following filtrate, i.e. the saturated solution, was used in the test. During filtration the filter was always kept covered.

Test concentration:
The saturated solution was tested in a limit test.

Control:
Six replicates (without test item) were tested under the same test conditions as the test replicates.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus CHODAT
- Strain: SAG 86.81
- Source (laboratory, culture collection):
Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): Four days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 35 - 70 µExm-2xs-1 for 24 h per day.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No

Test conditions

Hardness:

Dilution water: 0.24 mmol Ca+Mg/L

Test temperature:

min.: 22.0°C, max.: 23.0 °C, mean value: 22.5 °C

pH:

Nominal test item concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
1.00 7.90 8.13
Control 8.15 8.34

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 Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at approximately 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 103 cells/mL, current: 4847 cells/mL
- Control end cells density: Mean 2048940 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water, composition acc. to the guideline
- Hardness: 0.24 mmol Ca+Mg/L
- pH-value: 8.1 +/- 0.2
- Culture medium different from test medium: Nutrient Medium Z acc. to LÜTTGE et al. (1994)

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
Cell density was measured daily via Chlorophyll-a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal. Self-fluorescence were checked in the definitive test. No Self-fluorescence was found.

TEST CONCENTRATIONS
- Range finding study: No

Reference substance (positive control):

yes

Remarks:

Potassium dichtromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

Results with reference substance valid? Yes

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 0.546 0.671 ± 0.413
95 % confidence interval 0.496 - 0.606
Yield Inhibition
EyC50 0.281 0.317 ± 0.139
95 % confidence interval 0.220 - 0.347
SD = Standard deviation

Reported statistics and error estimates:

EC-values and statistical analyses
EC-values of the growth rate and yield inhibition were estimated empirically based on the results of the only treatment level (Limit test design).
NOEC and LOEC values
NOEC/LOEC were determined by calculation of statistical significance of growth rate and yield. As standard a t-test was used for NOEC/LOEC calculations. When running a t-test a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance tests are 0.05. The alpha-value (acceptable probability of incorrectly concluding that there is a difference) is alpha=0.05.

Any other information on results incl. tables

Cell Densities

Nominal test item concentration	Replicate	Cell density [cells/mL]
[mg/L]	No.	0 hours	24 hours	48 hours	72 hours
1.00	1	4847	52177	356158	1979990
	2	4847	51206	309640	1813162
	3	4847	45034	261112	1419182
	4	4847	55549	280998	1912451
	5	4847	57368	342794	2215223
	6	4847	50878	290542	1895902
	Mean	4847	52035	306874	1872652
Control	1	4847	65295	353156	2228308
	2	4847	60168	379594	1930442
	3	4847	56651	324370	1807004
	4	4847	63833	368444	2045605
	5	4847	57147	378045	2447859
	6	4847	51696	347980	1834424
	Mean	4847	59132	358598	2048940

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 concentration	Replicate	Growth rate		Inhibition of growth rate	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
1.00	1		2.00	0		1975143	3
	2		1.98	2		1808315	12
	3		1.89	6		1414335	31
	4		1.99	1		1907604	7
	5		2.04	-1		2210376	-8
	6		1.99	1		1891055	7
	Mean	(-)	1.98	2	(-)	1867805	9
Control	1		2.04			2223461
	2		2.00			1925595
	3		1.97			1802157
	4		2.02			2040758
	5		2.08			2443012
	6		1.98			1829577
	Mean		2.01			2044093

Section-by-Section and Average Specific Growth Ratesof the Control Group (0 – 72 hours)

 

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 h)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	2.60	1.69	1.84	2.04	0.489	23.9	21.2
	2	2.52	1.84	1.63	2.00	0.466	23.3
	3	2.46	1.75	1.72	1.97	0.420	21.3
	4	2.58	1.75	1.71	2.02	0.488	24.2
	5	2.47	1.89	1.87	2.08	0.340	16.4
	6	2.37	1.91	1.66	1.98	0.358	18.1
				Mean	2.01
				SD ±	0.04
				CV [%]	1.96

CV= Coefficient of variation

SD= Standard deviation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study the test item was found not to inhibit the growth rate of the freshwater green alga Desmodesmus subspicatus after 72 hours. The NOEC-value of the test item for both inhibition of growth rate and inhibition of yield after 72 hours was 1.00 mg/L. The EC50-values of the test item for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were both > 1.00 mg/L, respectively.
All effect levels are given based on the saturated solution prepared with a nominal concentration of 1 mg/L of the test item.

Executive summary:

The toxicity of the test item to the unicellular freshwater green alga Desmodesmus subspicatuswas determined according to the principles of OECD 201 (2011) and Council Regulation (EC) No.761/2009/Method C.3 (2008). The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 4847 cells/mL. A saturated solution with a nominal concentration of 1.00 mg/L was tested. Six replicates were tested for the limit concentration and the control. Environmental conditions were determined to be within the acceptable limits.

No analytical determination was carried out since no suitable method for the determination of the test item could be established. All effect values of the test item are given based onthe nominal concentration.

NOEC, LOEC, EC - values of the test item (0 -72 hours)

based on the saturated solution, prepared with 1 mg/L

                               

	Growth Rate Inhibition Nominal test item concentration [mg/L]
NOEC	1.00
LOEC	> 1.00
ErC10	> 1.00
ErC20	> 1.00
ErC50	> 1.00
	Inhibition of Yield Nominal test item concentration [mg/L]
NOEC	1.00
LOEC	> 1.00
EyC10	> 1.00
EyC20	> 1.00
EyC50	> 1.00