Hydrogen bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-), compound with 2-ethylhexylamine (1:1)

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:

from 2017-09-09 to 2017-09-14, with the definitive exposure phase from 2017-09-11 to 2017-09-14

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:

Hydrogen bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-), compound with 2-ethylhexylamine (1:1)

EC Number:

275-864-7

EC Name:

Hydrogen bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-), compound with 2-ethylhexylamine (1:1)

Cas Number:

71701-15-0

Molecular formula:

C42H44CrN9O6

IUPAC Name:

Chromate(1-), bis[2-[2-[4,5-dihydro-3-methyl-5-(oxo-kO)-1-phenyl-1H-pyrazol-4-yl]diazenyl-kN1]benzoato(2-)-kO]-, hydrogen, compd. with 2-ethyl-1-hexanamine (1:1:1)

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 Savinyl Yellow RLS 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.

Test solutions

Vehicle:

no

Details on test solutions:

Preparation of the saturated solution
The saturated solution (100 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 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 concentrations (nominal)
Five dilution levels of the saturated solution were tested in a geometrical series with a dilution factor of 3.16: 0.100 - 0.316 - 1.00 - 3.16 - 10.0 % of the saturated solution. The dilution levels are based on the results of a preliminary range finding test (non-GLP, Erlenmeyer flasks, with headspace in OECD TG 201 medium).

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): 3 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:

No

Post exposure observation period:

No

Test conditions

Hardness:

Not specified

Test temperature:

min.: 21.5 max.: 22.5 mean value: 22.0

pH:

% of the saturated solution Geometric mean measured test item concentration pH-values
[mg/L] Start; 0 hours End; 72 hours
10.0 1.22 7.91 7.87
3.16 0.230 7.96 7.92
1.00 0.113 8.01 8.06
0.316 0.0750 8.05 8.15
0.100 0.0750 8.07 8.27
Control 8.17 8.32

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

nominal: 0.100 - 0.316 - 1.00 - 3.16 - 10.0% of the stock solution
geometric mean measured test item concentrations 0.0750 - 0.0750 - 0.113 - 0.230 - 1.22 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 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 10^3 cells/mL, Actual: mean 5836 cells/mL
- Control end cells density: Mean 1047556 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: 6140 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 concentration 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. The adsorption of the test item at the relevant wavelength (436 nm) was considered not relevant for the measurement of the fluorescence.

Reference substance (positive control):

yes

Remarks:

(Potassium dichromate)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.113 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.:

0.23 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.:

0.132 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI: 0.126 - 0.138

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.169 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI: 0.162 - 0.175

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.353 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI: 0.341 - 0.366

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.113 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.:

0.23 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.:

< 0.113 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 9

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.121 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: CI: 0.113 - 0.220

Effect concentrations 10

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.155 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: CI: 0.114 - 0.224

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 2017-10-09 to 2017-10-12. 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 0.611 0.665 ± 0.368
95% confidence interval (0.582 – 0.645)
Yield Inhibition
EyC50 0.271 0.316 ± 0.130
95% confidence interval (0.262 – 0.280)
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 statistical analyses
NOEC/LOEC was determined by calculation of statistical significance of growth rates and yield. As standard One Way Analysis of Variance (ANOVA) and DUNNETT’s test was used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 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.
• GraphPad Prism, GRAPHPAD SOFTWARE, INC.

Any other information on results incl. tables

Cell Densities

% of the Saturated solution	Geometric mean measured test item concentration	Replicate	Cell density [cells/mL]
[%]	[mg/L]	No.	0 hours	24 hours	48 hours	72 hours
10.0	1.22	1	5836	10664	8614	4575
		2	5836	7730	5228	6086
		3	5836	8132	4811	5498
		Mean	5836	8842	6218	5386
3.16	0.230	1	5836	22534	70785	165450
		2	5836	15717	67399	180163
		3	5836	19672	70493	181499
		Mean	5836	19308	69559	175704
1.00	0.113	1	5836	27511	184229	945566
		2	5836	27553	158511	826447
		3	5836	30145	187892	966596
		Mean	5836	28403	176877	912870
0.316	0.0750	1	5836	24284	154031	739284
		2	5836	28217	185348	847849
		3	5836	29561	192743	983594
		Mean	5836	27354	177374	856909
0.100	0.0750	1	5836	27864	192747	909834
		2	5836	31588	211924	1024435
		3	5836	28338	154973	748884
		Mean	5836	29263	186548	894384
Control		1	5836	28820	202328	1094582
		2	5836	22177	164011	1074804
		3	5836	35486	147521	1112954
		4	5836	29769	151351	840022
		5	5836	23062	195214	985295
		6	5836	24592	195085	1177676
		Mean	5836	27318	175918	1047556

 

  Evaluation after 72 hours

                  Statistically significant differences of growth rates and yield compared to

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

 

% of the Saturated solution	Geometric mean measured test item concentration	Replicate	Growth rate		Inhibition of growth rate	Yield		Inhibition of yield
[%]	[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
10.0	1.22	1		-0.0811	100		-1261	100
		2		0.0140	99		250	100
		3		-0.0199	100		-338	100
		Mean	(+)	-0.0290	100	(+)	-450	100
3.16	0.230	1		1.12	35		159614	85
		2		1.14	34		174327	83
		3		1.15	34		175663	83
		Mean	(+)	1.14	34	(+)	169868	84
1.00	0.113	1		1.70	2		939730	10
		2		1.65	4		820611	21
		3		1.70	1		960760	8
		Mean	(-)	1.68	3	(-)	907034	13
0.316	0.0750	1		1.61	7		733448	30
		2		1.66	4		842013	19
		3		1.71	1		977758	6
		Mean	(-)	1.66	4	(-)	851073	18
0.100	0.0750	1		1.68	3		903998	13
		2		1.72	0		1018599	2
		3		1.62	6		743048	29
		Mean	(-)	1.68	3	(-)	888548	15
Control		1		1.75			1088746
		2		1.74			1068968
		3		1.75			1107118
		4		1.66			834186
		5		1.71			979459
		6		1.77			1171840
		Mean		1.73			1041720

 

 

 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	1.60	1.95	1.69	1.75	0.183	10.5	15.3
	2	1.34	2.00	1.88	1.74	0.355	20.4
	3	1.81	1.43	2.02	1.75	0.302	17.2
	4	1.63	1.63	1.71	1.66	0.0497	3.00
	5	1.37	2.14	1.62	1.71	0.389	22.7
	6	1.44	2.07	1.80	1.77	0.317	17.9
				Mean	1.73
				SD ±	0.04
				CV [%]	2.32

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 0.353 (0.341 – 0.366) and 0.155 (0.114 – 0.224) mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 0.113 mg/L, respectively. All effect levels are given based on the geometric mean measured test item 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) 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 100 mg/L was prepared in demineralized water two days prior to the start of exposure. The dispersion was stirred with approximately 1100 rpm for 48 hours at room temperature with a magnetic stirrer. Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC,Macherey-Nagel). The saturated solution and four dilution levels of the saturated solution were tested in a geometrical series with a dilution factor of √10:0.100 - 0.316 - 1.00 - 3.16 - 10.0% of the saturated solution, corresponding to the geometric mean measured test item concentrations 0.0750 – 0.0750 – 0.113 – 0.230 – 1.22 mg/L (the two lowest concentration lowest were < LOQ).Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditionswere within the acceptable limits.

 

The test media were colored yellow in a concentration-related manner. The concentrations of the test item Savinyl Yellow RLS 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 < LOQ to 2.52 mg/L. The measured concentrations in the old media (72 h) were in the range from < LOQ to 0.589 mg/L.

 

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	0.113
LOEC	0.230
ErC10	0.132 (0.126 - 0.138)
ErC20	0.169 (0.162 - 0.175)
ErC50	0.353 (0.341 - 0.366)
	Inhibition of Yield
NOEC	0.113
LOEC	0.230
EyC10	< 0.113
EyC20	0.121 (0.113 – 0.220)
EyC50	0.155 (0.114 – 0.224)