Sodium bis[4-hydroxy-3-[(2-hydroxy-1-naphthyl)azo]-N-(3-methoxypropyl)benzenesulphonamidato(2-)]cobaltate(1-)

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 - 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 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:

Sodium bis[4-hydroxy-3-[(2-hydroxy-1-naphthyl)azo]-N-(3-methoxypropyl)benzenesulphonamidato(2-)]cobaltate(1-)

EC Number:

275-959-3

EC Name:

Sodium bis[4-hydroxy-3-[(2-hydroxy-1-naphthyl)azo]-N-(3-methoxypropyl)benzenesulphonamidato(2-)]cobaltate(1-)

Cas Number:

71735-61-0

Molecular formula:

C40H38CoN6O10S2.Na

IUPAC Name:

Cobaltate(1-), bis[4-(hydroxy-kO)-3-[2-[2-(hydroxy-kO)-1-naphthalenyl]diazenyl-kN1]-N-(3-methoxypropyl)benzenesulfonamidato(2-)]-, sodium (1:1)

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
Analytical evaluation of the saturated solution of the test item and the control was carried out via HPLC-DAD at the start (0 hours) and at the end of the exposure (72 hours) with algae. Additionally, the saturated solution was analysed before start of the exposure for calculation of the dilution levels.

Test solutions

Vehicle:

no

Details on test solutions:

Preparation of the saturated solution
The saturated solution (120 mg/L test item was weighed out) was prepared with demineralized water four days prior to the start of the exposure (0 h).
The dispersion was stirred with a magnetic stirrer with approximately 900 – 1000 rpm for 72 hours at 30 °C and 24 hours at room temperature and protected from light. 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
5 concentrations (saturated solution and dilutions) were tested in a geometrical series with a dilution factor of 2.2: 5.14 – 11.3 – 24.8 – 54.5 – 120 mg/L, corresponding to the geometric mean measured test item concentrations 4.20 – 11.1 – 24.2 – 58.- - 98.7 mg/L. The concentration levels are based on the results of preliminary range finding tests (non-GLP, Erlenmeyer flasks, with headspace).

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 Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 2590 - 5180 lux corresponding to 35 - 70 µE ∙ m-2 ∙ s-1 for 24 h per day.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

The method was modified according to OECD series,no.23. The saturated solution was red. Since this may affect the light intensities for the algae cultures volumes of test media in the test vessels were lowered to minimize the water layer and path length.

Post exposure observation period:

No

Test conditions

Hardness:

Not specified

Test temperature:

min.: 22.0 max.: 22.5 mean value: 22.3

pH:

Geometric mean measured test item concentration pH-values
[mg/L] Start; 0 hours End; 72 hours
98.7 7.75 7.83
58.2 7.80 7.99
24.2 7.87 8.05
11.1 7.90 8.09
4.20 8.06 8.29
Control 8.22 8.56

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

Nominal: 5.14 - 11.3 - 24.8 - 54.5 - 120 mg/L
geometric mean measured test item concentrations: 4.20 - 11.1 – 24.2 – 58.2 – 98.7 mg/L.

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 25 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 10^3 cells/mL, Actual: mean 5836 cells/mL
- Control end cells density: Mean 1454547 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Composition of Dilution Water
according to the guideline
Component Concentration [mg/L]
NH4Cl 15
MgCl2 x 6 H2O 12
CaCl2 x 2 H2O 18
MgSO4 x 7 H2O 15
KH2PO4 1.6
FeCl3 x 6 H2O 0.064
Na2EDTA x 2 H2O 0.1
H3BO3 0.185
MnCl2 x 4 H2O 0.415
ZnCl2 3 x 10^-3
Na2MoO4 x 2 H2O 7 x 10^-3
CoCl2 x 6 H2O 1.5 x 10^-3
CuCl2 x 2 H2O 1 x 10^-5
NaHCO3 50
pH 8.1 ± 0.2



OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120
µE ∙ m-2 ∙ s-1, mean value: 5576 lux,



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
-The 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 was observed in the definitive test. A separate replicate for the loading levels were prepared without algae and treated like the other test replicates. The self-fluorescence of each measuring point was taken into account in the evaluation.

Reference substance (positive control):

yes

Remarks:

(Potassium dichromate)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

11.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

24.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

29.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI: 24.4 - 34.9

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

42.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI: 37.9 - 47.1

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

90 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI: 82.8 - > 98.7

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

11.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 7

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

24.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 8

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

15.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: CI: 7.33 - 21.9

Effect concentrations 9

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

19.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: CI: 13.9 - 26.0

Effect concentrations 10

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

32.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: CI: 25.6 - 44.3

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
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

The toxicity of potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) to the unicellular freshwater green alga Desmodesmus subspicatus was determined over a period of 72 hours from 2017-04-10 to 2017-04-13. The reference item toxicity is in the valid range following test facility SOPS.

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 0.697 0.668 ± 0.375
95% confidence interval 0.647 – 0.755
Yield Inhibition
EyC50 0.325 0.318 ± 0.130
95% confidence interval 0.308 – 0.343
SD = Standard deviation

Reported statistics and error estimates:

EC-values and statistical analyses
EC10-, EC20- and EC50-value with confidence intervals of yield inhibition after 72 hours was calculated by sigmoidal dose-response regression.

NOEC, LOEC and analyses
The NOEC / LOEC was determined by calculation of statistical significance of growth rate and yield.
The following statistical tests were conducted:

Shapiro-Wilk’s test on normal distribution was done with a significance level 0.01 for growth rate and yield.
Levene’s test on variance homogeneity was done with a significance level 0.01 for growth rate and yield.
Monotonicity was done by trend analysis by contrasts (significance level 0.05) for growth rate and yield.
William’s test on significant differences was done with a significance level 0.05 for growth rate and yield.

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.
• GraphPad Prism, GRAPHPAD SOFTWARE, INC.
• ToxRat Professional, Version 3.2.1, TOXRAT SOLUTIONS GMBH

Any other information on results incl. tables

 Cell Densities

Geometric mean measured test item concentration	Replicate	Cell density [cells/mL]
[mg/L]	No.	0 hours	24 hours	48 hours	72 hours
98.7	1	5836	11681	25655	67441
	2	5836	9180	27701	83927
	3	5836	12080	22519	76794
	Mean	5836	10980	25292	76054
58.2	1	5836	19103	62768	291249
	2	5836	15216	55108	207236
	3	5836	18252	65068	248484
	Mean	5836	17524	60981	248990
24.2	1	5836	24527	124301	875275
	2	5836	26607	151727	964064
	3	5836	28562	146250	1180815
	Mean	5836	26565	140759	1006718
11.1	1	5836	29063	200422	1414539
	2	5836	34810	214217	1442629
	3	5836	35380	178702	1392826
	Mean	5836	33084	197780	1416665
4.20	1	5836	32654	168247	1515865
	2	5836	27928	192739	1108099
	3	5836	36796	236082	1904006
	Mean	5836	32459	199023	1509323
Control	1	5836	44756	302258	1701850
	2	5836	36572	292168	1997179
	3	5836	44069	161426	1199640
	4	5836	43484	207229	1279925
	5	5836	44942	229686	1263109
	6	5836	49144	262240	1285581
	Mean	5836	43828	242501	1454547

 

 Evaluation after 72 hours

 Statistically significant differences of growth rates and yield compared to

 control values are marked (+), not significant differences are marked (-).

 

Geometric mean measured test item concentration	Replicate	Growth rate		Inhibition of growth rate	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
98.7	1		0.816	56		61605	96
	2		0.889	52		78091	95
	3		0.859	53		70958	95
	Mean	(+)	0.854	53	(+)	70218	95
58.2	1		1.30	29		285413	80
	2		1.19	35		201400	86
	3		1.25	32		242648	83
	Mean	(+)	1.25	32	(+)	243154	83
24.2	1		1.67	9		869439	40
	2		1.70	7		958228	34
	3		1.77	3		1174979	19
	Mean	(+)	1.71	7	(+)	1000882	31
11.1	1		1.83	0		1408703	3
	2		1.84	0		1436793	1
	3		1.83	0		1386990	4
	Mean	(-)	1.83	0	(-)	1410829	3
4.20	1		1.85	-1		1510029	-4
	2		1.75	5		1102263	24
	3		1.93	-5		1898170	-31
	Mean	(-)	1.84	-1	(-)	1503487	-4
Control	1		1.89			1696014
	2		1.95			1991343
	3		1.78			1193804
	4		1.80			1274089
	5		1.79			1257273
	6		1.80			1279745
	Mean		1.83			1448711

 

 

 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	2.04	1.91	1.73	1.89	0.155	8.21	13.1
	2	1.84	2.08	1.92	1.95	0.123	6.32
	3	2.02	1.30	2.01	1.78	0.413	23.3
	4	2.01	1.56	1.82	1.80	0.224	12.5
	5	2.04	1.63	1.71	1.79	0.219	12.2
	6	2.13	1.68	1.59	1.80	0.291	16.2
				Mean	1.83
				SD ±	0.07
				CV [%]	3.74

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 to inhibit the growth rate of the freshwater green alga Desmodesmus subspicatus after 72 hours with the following values: The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 90.0 (82.8 – > 98.7) and 32.2 (25.6 – 44.3) mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 11.1 mg/L, respectively.
All effect levels are given based on the geometric mean measured test item concentrations.

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) andCouncil Regulation (EC) No. 266/2016/Method C.3 (2016) . 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 5836 cells/mL. A saturated solution with a nominal concentration of 120 mg/L was prepared in demineralized water four days prior to the start of exposure. The dispersion was stirred with approximately 900 – 1000 rpm for 72 hours at 30 °C and 24 hours at room temperature and protected from light with a magnetic stirrer. Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC,Macherey-Nagel). The saturated solution and four concentrations out of the saturated solution were tested in a geometrical series with a dilution factor of 2.2:5.14 - 11.3 - 24.8 - 54.5 - 120 mg/L, corresponding to the geometric mean measured test item concentrations 4.20 - 11.1 – 24.2 – 58.2 – 98.7 mg/L.Three replicates were tested for each test item concentration and six replicates for the control.The environmental conditions were within the acceptable limits.

 

The test media were colored red in a concentration-related manner and after 24 hours adsorption to glass was observed. The concentrations of the test item were determined at the start of the exposure (0 hours) and at the end of the exposure (72 hours) in all test media and the control via HPLC-DAD. The measured concentrations of the test item at the start of the exposure (0 hours) ranged from 79% to 106% of the nominal test item concentrations.The measured concentrations in the old media (72 h) was 74 - 107% of the nominal values.

 

All effect values of the test item are given based on the geometric mean measured test item concentration.

 

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

                  based on geometric mean measured test item concentration [mg/L]

                               

            

	Growth Rate Inhibition
NOEC	11.1
LOEC	24.2
ErC10	29.7 (24.4 - 34.9)
ErC20	42.7 (37.9 - 47.1)
ErC50	90.0 (82.8 - > 98.7)
	Inhibition of Yield
NOEC	11.1
LOEC	24.2
EyC10	15.5 (7.33 – 21.9)
EyC20	19.8 (13.9 – 26.0)
EyC50	32.2 (25.6 – 44.3)