[2,3'-bis[[(2-hydroxyphenyl)methylene]amino]but-2-enedinitrilato(2-)-N2,N3,O2,O3]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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well performed OECD study with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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'-bis[[(2-hydroxyphenyl)methylene]amino]but-2-enedinitrilato(2-)-N2,N3,O2,O3]nickel

EC Number:

265-022-7

EC Name:

[2,3'-bis[[(2-hydroxyphenyl)methylene]amino]but-2-enedinitrilato(2-)-N2,N3,O2,O3]nickel

Cas Number:

64696-98-6

Molecular formula:

C18H10N4NiO2

IUPAC Name:

[2,3'-bis[[(2-hydroxyphenyl)methylene]amino]but-2-enedinitrilato(2-)-N2,N3,O2,O3]nickel

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
The test concentrations were analytically verified via HPLC-DAD in the saturated solution and the control at the start of the exposure (0 hours). A measured concentration below the LOQ was determined at the start of the exposure (0 hours). Therefore, no analytical monitoring at the end of the exposure was carried out and the test item concentrations was considered to be below the LOQ in the aged media, too.

Sampling for the analytical monitoring
At the start of the exposure (0 hours), sampling was carried out directly after preparation of the saturated solution.

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 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): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 35 - 70 µE x m-2 x s-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 max.: 22.0, mean value: 22.0

pH:

Nominal test item concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
1.00 7.83 8.29
Control 8.00 8.43

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 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 103 cells/mL, Actual: mean 4847 cells/mL
- Control end cells density: Mean 880586 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: 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, mean value: 69.1



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] fluorimeter, Cell density and self fluorescence was measured daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal.



TEST CONCENTRATIONS
- Range finding study:No

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

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: 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

The toxicity of potassium dichromate as reference substance is regularly determined over a period of 72 hours. The toxicity data of the reference item are in the valid range following the SOP of the test facility.


EC50-Values of the Recent Studies with the Reference Substance Potassium Dichromate
based on nominal concentrations mg/L, (0-72 hours)
Reference Substance Potassium Dichromate, CAS RN 7778-50-9, Sigma-Aldrich,
batch number MKBQ9179V, purity 100.0 %


Growth Rate Inhibition Valid Range (average ± 3 x SD)
ErC50 0.685 0.671 ± 0.401
95 % confidence interval 0.603 - 0.794
Yield Inhibition
EyC50 0.362 0.320 ± 0.139
95 % confidence interval 0.328 - 0.398

SD = Standard deviation

Reported statistics and error estimates:

EC-values and statistical analyses
EC-values of 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 by t-test analysis. When running a t-test, a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance tests were 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is  = 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.

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	24137	154516	892576
	2	4847	25739	232744	1255144
	3	4847	28115	175312	1032225
	4	4847	27923	179814	1116024
	5	4847	27326	254868	1103998
	6	4847	25720	166181	1000380
	Mean	4847	26493	193906	1066725
Control	1	4847	21775	132907	749127
	2	4847	22848	130574	827442
	3	4847	26354	156632	831940
	4	4847	23723	160101	1130504
	5	4847	27836	150764	683945
	6	4847	23507	181724	1060559
	Mean	4847	24341	152117	880586

 

 

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		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
1.00	1		1.74	-1		887729	-1
	2		1.85	-7		1250297	-43
	3		1.79	-3		1027378	-17
	4		1.81	-5		1111177	-27
	5		1.81	-5		1099151	-26
	6		1.78	-3		995533	-14
	Mean	(-)	1.80	-4	(-)	1061878	-21
Control	1		1.68			744280
	2		1.71			822595
	3		1.72			827093
	4		1.82			1125657
	5		1.65			679098
	6		1.80			1055712
	Mean		1.73			875739

 

 

 

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	1.50	1.81	1.73	1.68	0.159	9.46	8.87
	2	1.55	1.74	1.85	1.71	0.150	8.77
	3	1.69	1.78	1.67	1.72	0.059	3.46
	4	1.59	1.91	1.96	1.82	0.200	11.0
	5	1.75	1.69	1.51	1.65	0.123	7.44
	6	1.58	2.05	1.76	1.80	0.235	13.1
				Mean	1.73
				SD ±	0.07
				CV [%]	3.78

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-values of the test item for both inhibition of growth rate and inhibition of yield after 72 hours were 1.00 mg/L, respectively. The EC50-values of the test item for both inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 1.00 mg/L, respectively.
All effect levels are given based on the nominal concentration of the test item.

Executive summary:

The toxicity of the test item to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 (2011) and Council Regulation (EC)761/2009/Method C.3 . 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 assessed. Six replicates were tested for the limit concentration and the control. Environmental conditions were determined to be within the acceptable limits.

The test item concentration in the test and control solutions were analysed by HPLC-DAD at the start of the exposure. The measured concentration of the test item was below the SysQL. Therefore, no analytical monitoring was carried out at the end of the exposure. The test item concentration at the end of the exposure is considered to be below the SysQL.

All effect values of Polysynthren-Braun R are given based on the nominal concentration.

 

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

based on nominal test item concentrations [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