Calcium bis[4-[[3-[[2-hydroxy-3-[[(4-methoxyphenyl)amino]carbonyl]-1-naphthyl]azo]-4-methylbenzoyl]amino]benzenesulphonate]

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2016-09-10 to 2017-04-21, with the definitive exposure phase from 2017-04-18 to 2017-04-21

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
The test concentration of Pigment Red 247 was analytically verified via HPLC-DAD in the saturated solution and the control at the start and the end of the exposure.

Vehicle:

no

Details on test solutions:

Preparation of the saturated solution
The saturated solution (10.0 mg/L test item were weighed out) was prepared with dilution water.
The dispersion was stirred for 15 minutes (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. During filtration the filter was always kept covered. The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item, which was negative. The following filtrate, i.e. the saturated solution, was used in the test.

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


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

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 4996 – 6412 lux corresponding to 35 - 70 µE ∙ m-2 ∙ s-1 for 24 h per day.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No

Hardness:

Not specified

Test temperature:

min.: 22.0 max.: 23.0 mean value: 22.5

pH:

Geometric mean measured test item concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
0.243 8.05 9.11
Control 8.21 9.15

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

Nominal: A saturated solution with a nominal concentration of 10.0 mg/L
Measured: 0.242 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 6839 cells/mL
- Control end cells density: Mean 2242572 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:
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 4996 to 6412 lux, corresponding to 60 to 120
µE ∙ m-2 ∙ s-1, mean value: 5787 lux,



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
-Chlorophyll a-fluorescence
The 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 range finding test. A separate replicate for the loading levels were prepared without algae and treated the same as 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)

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.243 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 0.243 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.243 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 0.243 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.243 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.243 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 0.243 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.243 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 0.243 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.243 mg/L

Nominal / measured:

meas. (geom. mean)

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):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: The limit concentration was visually clear and slightly pink, throughout the exposure 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.

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 0.773 ± 0.540
95% confidence interval 0.538 – 0.581
Yield inhibition
EyC50 0.320 0.406 ± 0.287
95% confidence interval 0.242 – 0.341
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 a 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 normal distribution of data. The Levene median test was used for equal variance. 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 are 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.

Cell Densities

Geometric mean measured test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 hours		24 hours		48 hours		72 hours
0.243		1		6839		28845		276376		2111575
		2		6839		23280		214170		2010862
		3		6839		28664		306842		2284560
		4		6839		27965		271098		2215390
		5		6839		28161		280742		2139021
		6		6839		26420		254162		2297201
		Mean		6839		27223		267232		2176435
Control		1		6839		30739		302898		2590854
		2		6839		23845		243746		2322891
		3		6839		25823		216581		1963520
		4		6839		27368		279267		1942277
		5		6839		28374		303647		2441335
		6		6839		24661		257357		2194557
		Mean		6839		26802		267249		2242572

 

 

 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		Growth rate inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
0.243	1		1.91	1		2104736	6
	2		1.90	2		2004023	10
	3		1.94	0		2277721	-2
	4		1.93	0		2208551	1
	5		1.92	1		2132182	5
	6		1.94	-1		2290362	-2
	Mean	(-)	1.92	0	(-)	2169596	3
Control	1		1.98			2584015
	2		1.94			2316052
	3		1.89			1956681
	4		1.88			1935438
	5		1.96			2434496
	6		1.92			2187718
	Mean		1.93			2235733

 

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	1.50	2.29	2.15	1.98	0.418	21.1	26.2
	2	1.25	2.33	2.25	1.94	0.602	31.0
	3	1.33	2.13	2.21	1.89	0.485	25.7
	4	1.39	2.32	1.94	1.88	0.471	25.0
	5	1.42	2.37	2.08	1.96	0.486	24.8
	6	1.28	2.35	2.14	1.92	0.564	29.3
				Mean	1.93
				SD ±	0.04
				CV [%]	2.01

SD = Standard deviation    CV = Coefficient of variation

Validity criteria fulfilled:

yes

Conclusions:

In this study, no inhibition of growth on the freshwater green alga Pseudokirchneriella subcapitata was found after 72 hours exposure to the test item Pigment Red 247. The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 0.243 mg/L, respectively. This concentration refers to a saturated solution with a nominal loading of 10 mg test item /L. The NOEC-values for inhibition of growth rate and yield after 72 hours were 0.243 mg/L, respectively.
All effect values given are based on geometric mean measured test item concentrations of Pigment Red 247.

Executive summary:

The toxicity of Pigment Red 247 (batch number: MA566.004) 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 .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 saturated solutionwith a nominal loading of 10.0 mg test item/L was tested. Sixreplicates were used for the limit loading level and the control. The environmental conditionswere within the acceptable limits.

 

The concentration of the test item Pigment Red 247 was analytically verified via HPLC-DAD at the start of the exposure (0 hours) and at the end of the exposure (72 hours) in the limit concentration and the control.

The measured concentration of the test item at the start of the exposure (0 hours) was 0.270 mg/L. The measured concentrations at the end of the exposure (72 hours) was 0.218 mg/L. The geometric mean measured concentration was 0.243 mg/L.

 

All effect values were based on the geometric mean measured test item concentration of the test item Pigment Red 247. The results are summarized in the following table:

 

NOEC, LOEC, EC - valuesofPigmentRed247(0-72hours)

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

 

	Inhibition of Growth Rate
NOEC	0.243
LOEC	>0.243
ErC10	>0.243
ErC20	>0.243
ErC50	>0.243
	Inhibition of Yield
NOEC	0.243
LOEC	>0.243
EyC10	>0.243
EyC20	>0.243
EyC50	>0.243

 

Description of key information

The toxicity of Pigment Red 247 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 .The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours.

In this study, no inhibition of growth on the freshwater green alga Pseudokirchneriella subcapitata was found after 72 hours exposure to the test item Pigment Red 247. The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 0.243 mg/L, respectively. This concentration refers to a saturated solution with a nominal loading of 10 mg test item /L. The NOEC-values for inhibition of growth rate and yield after 72 hours were 0.243 mg/L, respectively.