Disodium [5-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxybenzene-1,3-disulphonato(4-)]cuprate(2-)

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:

2017-09-25 to 2017-09-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

according to OECD 201 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 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:

Disodium [5-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxybenzene-1,3-disulphonato(4-)]cuprate(2-)

EC Number:

276-541-3

EC Name:

Disodium [5-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxybenzene-1,3-disulphonato(4-)]cuprate(2-)

Cas Number:

72252-61-0

Molecular formula:

C16H10CuN4O8S2.2Na

IUPAC Name:

disodium [5-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxybenzene-1,3-disulphonato(4-)]cuprate(2-)

Test material form:

solid: nanoform

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
All concentration levels and the control were analytically verified via HPLC-DAD at the start (0 hours) and at the end of the exposure (72 hours) with algae.

Test solutions

Vehicle:

no

Details on test solutions:

Stock solution, media preparation
100 mg main component/L, corresponding to 124.4 mg test item/L were freshly prepared with dilution water. The dispersion was agitated until the stock solution was clear.

Test concentrations (nominal)
Five concentration levels were tested in a geometrical series with a dilution factor of 2: 6.25 - 12.5 - 25.0 - 50.0 - 100 mg main component/L, corresponding to 7.77 - 15.5 - 31.1 - 62.2 - 124.4 mg test item/L. The concentration levels are based on the results of a preliminary range finding test.

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

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDÁK
- Strain: SAG 61.81
- Source (laboratory, culture collection): SAG, Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 2567 – 5130 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 acc. to OECD no. 23. The test item solutions are orange. Since this may affect the light intensities for algae cultures volumes of test media in the test vessels were lowered in order to minimize the water layer and path length.

Post exposure observation period:

No

Test conditions

Hardness:

Not specified

Test temperature:

min.: 21.8 max.: 22.5 mean value: 22.2

pH:

Nominal main component concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
100 7.57 8.00
50.0 7.63 8.06
25.0 7.72 8.33
12.5 7.80 8.50
6.25 7.88 8.70
Control 8.10 8.91

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 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 6839 cells/mL
- Control end cells density: Mean 1914404 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 0.064a2EDTA 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: 5597 lux,



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
-Chlorophyll a-fluorescence
Cell density was measured daily via Chlorophyll a fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. Self-fluorescence was observed in the preliminary range finding test at the concentration of 100 mg test item/L. A separate replicate of each test item concentration was 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.:

31.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

62.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 124.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

15.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 5

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

31.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

59.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: CI: 52.7 - 68.3

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: The test media were clearly dissolved and concentration-related orange colored throughout the test period.
- 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 Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2017-04-18 to 2017-04-21 (with headspace). The reference item toxicity is in the valid range following test facility SOPS.

Table 10: 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 0.559 0.754 ± 0.518
95% confidence interval 0.538 – 0.581
Yield inhibition
EyC50 0.320 0.398 ± 0.291
95% confidence interval 0.242 – 0.341
SD = Standard deviation

Reported statistics and error estimates:

EC-values and statistical analyses
EC10-, EC20- and EC50-values with confidence intervals of yield inhibition after 72 hours were 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 of 0.01.
Levene’s test on variance homogeneity was done with a significance level of 0.01.
Monotonicity of Concentration/Response was assessed by trend analysis by contrasts (significance level: 0.05) for yield.
Williams Multiple t-test was done with a significance level of 0.05 for yield.
Welsh-t-test after Bonferroni-Holm was done with a significance level of 0.05 for growth rate.

Software
The data for the tables in this report were computer-generated and have been rounded for presentation from the fully 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

Nominal main component concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 hours		24 hours		48 hours		72 hours
100		1		6839		5900		28091		700918
		2		6839		5830		22563		723624
		3		6839		5098		15248		413878
		Mean		6839		5609		21967		612807
50.0		1		6839		10037		59554		1158293
		2		6839		9715		62237		1559565
		3		6839		13282		72534		1292889
		Mean		6839		11011		64775		1336916
25.0		1		6839		23307		209909		1999784
		2		6839		24178		227307		2325097
		3		6839		23128		206615		2349793
		Mean		6839		23538		214610		2224891
12.5		1		6839		29261		266671		2818558
		2		6839		25674		252907		2781105
		3		6839		29837		227527		2714334
		Mean		6839		28257		249035		2771332
6.25		1		6839		34174		321524		2779130
		2		6839		35926		327564		2911269
		3		6839		34964		331953		2828580
		Mean		6839		35021		327014		2839660
Control		1		6839		27277		282620		2698033
		2		6839		37518		260640		2742801
		3		6839		33548		295588		2742626
		4		6839		40005		342791		2622718
		5		6839		31909		386868		2791197
		6		6839		41977		337465		2990809
		Mean		6839		35372		317662		2764697

 

 

 

 Evaluation after 72 hours

 Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

Nominal main component concentration		Replicate		Growth rate			Inhibition of growth rate		Yield			Inhibition of yield
[mg/L]		No.		[d^-1]			[%]		[cells/mL]			[%]
100		1				1.54		23				694079		75
		2				1.55		22				716785		74
		3				1.37		32				407039		85
		Mean		(+)		1.49		26		(+)		605968		78
50.0		1				1.71		14				1151454		58
		2				1.81		10				1552726		44
		3				1.75		13				1286050		53
		Mean		(+)		1.76		12		(+)		1330077		52
25.0		1				1.89		5				1992945		28
		2				1.94		3				2318258		16
		3				1.95		3				2342954		15
		Mean		(-)		1.93		4		(+)		2218052		20
12.5		1				2.01		0				2811719		-2
		2				2.00		0				2774266		-1
		3				1.99		0				2707495		2
		Mean		(-)		2.00		0		(-)		2764493		0
6.25		1				2.00		0				2772291		-1
		2				2.02		-1				2904430		-5
		3				2.01		0				2821741		-2
		Mean		(-)		2.01		0		(-)		2832821		-3
Control		1				1.99						2691194
		2				2.00						2735962
		3				2.00						2735787
		4				1.98						2615879
		5				2.00						2784358
		6				2.03						2983970
		Mean				2.00						2757858

 

 

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

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 hours)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.38	2.34	2.26	1.99	0.529	26.6	17.3
	2	1.70	1.94	2.35	2.00	0.330	16.5
	3	1.59	2.18	2.23	2.00	0.354	17.7
	4	1.77	2.15	2.04	1.98	0.196	9.89
	5	1.54	2.50	1.98	2.00	0.478	23.9
	6	1.81	2.08	2.18	2.03	0.190	9.39
				Mean	2.00
				SD ±	0.01
				CV [%]	0.74

SD = Standard deviation CV = Coefficient of variation

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, the test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on nominal main component concentrations): The EC50-values with 95% confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 100 mg/L and 47.9 (42.3 – 54.9) mg/L, respectively. The NOEC-values for inhibition of growth rate and yield after 72 hours were 25.0 and 12.5 mg/L, respectively.
All effect values given are based on nominal main component concentrations of the test item.

Executive summary:

The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3 from 2017-09-25 to 2017-09-28 at the test facility.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 6839 cells/mL. A stock solution with a nominal loading of 124.4 mg test item/L, corresponding to 100 mg main component/L was tested. Five concentrations were tested in a geometrical series with a dilution factor of 2 (nominal):6.25 - 12.5 - 25.0 - 50.0 - 100 mg main component/L, corresponding to 7.77 – 15.5 – 31.1 – 62.2 – 124.4 mg test item/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 concentration of the test item Sanodure Fast Gold L dried was analytically verified via HPLC-DAD at the start of the exposure (0 hours) and at the end of the exposure (72 hours) in all tested concentrations and the control.

The measured concentration of the test item at the start of the exposure (0 hours) were between 96 and 100% of the nominal values. The measured concentrations at the end of the exposure (72 hours) were between 89 and 104% of the nominal values.

 

All effect values were based on the nominal test item and main component concentration of the test item. The results are summarized in the following table:

 

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

based on the nominal main component and test item concentration [mg/L]

 

	Nominal test item concentration [mg/L]	Nominal main component concentration [mg/L]
	Inhibition of Growth Rate
NOEC	31.1	25.0
LOEC	62.2	50.0
ErC10	54.3 (44.7 – 66.9)	43.7 (35.9 – 53.8)
ErC20	93.0 (75.7 - 110)	74.8 (60.9 – 88.2)
ErC50	> 124.4	> 100
	Inhibition of Yield
NOEC	15.5	12.5
LOEC	31.1	25.0
EyC10	24.0 (19.3 – 29.1)	19.3 (15.6 – 23.4)
EyC20	32.2 (27.2 – 36.9)	25.9 (21.9 – 29.7)
EyC50	59.6 (52.7 – 68.3)	47.9 (42.3 – 54.9)