Disodium 2,2'-(azodi-p-phenylene)bis[6-methylbenzothiazole-7-sulphonate]

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:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Commission Regulation (EU) No.2016/266

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

PRELIMINARY TESTThe analytical determination of the test substance concentrations was performed at the beginning and at the end of the test. The samples for analysis were taken from the highest (100 mg/L), the lowest (1 mg/L) test concentration and from the highest (100 mg/L) concentration without algae. The samples for analysis (0 hours) were prepared at the beginning of the test and immediately delivered in transport box to analytical laboratory. The samples were analyzed on the day of delivery. The samples for analysis at the end of the test (72 hours) were delivered to analytical laboratory immediately after the end of testing. The samples were analyzed on the day of delivery. All samples were stored at laboratory temperature.The analytical results showed that the test substance was stable in the test medium under the conditions of testing.LIMIT TEST The analytical determination of the test substance concentration was performed at the beginning and at the end of the test. The samples for analysis were taken from the stock solution (100 mg/L) with and without algae. The samples for analysis (0 hours) were prepared at the beginning of the test and immediately delivered in transport box to analytical laboratory. The samples were analyzed on the day of delivery. The samples for analysis at the end of the test (72 hours) were delivered to analytical laboratory immediately after the end of testing. The samples were analyzed on the day of delivery. All samples were stored at laboratory temperature.

Vehicle:

no

Details on test solutions:

BASIC STOCK SOLUTIONSThe four basic stock solutions were prepared according to following data. The solutions were kept in the dark at temperature of 4 ± 1 °C in fridge. The temperature was checked every day.Solution A: NH4Cl: 1.5 g MgCl2 · 6H2O: 1.2 g CaCl2 · 2H2O: 1.8 g MgSO4 · 7H2O: 1.5 g KH2PO4: 0.16 g water up to 1000 mL Solution B: FeCl3 · 6H2O: 64 mg Na2EDTA · 2H2O: 100 mg water up to 1000 mLSolution C: H3BO3: 185 mg MnCl2 · 4H2O: 415 mg ZnCl2: 3.0 mg CoCl2 · 6H2O: 1.5 mg CuCl2 · 2H2O: 0.01 mg Na2MoO4 · 2H2O: 7.0 mg water up to 1000 mL Solution D: NaHCO3: 50 g water up to 1000 mLPREPARATION OF THE TEST MEDIUMThe test medium was prepared by admixing 10 mL of basic stock solution A and 1 mL of basic stock solutions B, C, D in 1 000 mL deionized water.PREPARATION OF SOLUTIONS OF THE TEST SUBSTANCEThe stock solution of the test substance was prepared in the test medium. 50 mg of the test substance was weighed into 500 mL of the test medium for the preliminary and the limit test. The stock solution was ultrasonicated for 10 minutes. The concentrations of test solutions used in the preliminary test were obtained by dilution of the stock solution with test medium.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM - Common name: Desmodesmus subspicatus - Strain: Brinkmann 1953/SAG 86.81 - Source (laboratory, culture collection): collection of autotrophic organisms of The Botanic Institute of the Czech Academy of Science, Třeboň on date 27.5.2016. - Method of cultivation: The strain culture was preinoculated from the stock solution and cultivated in flasks with the test medium on indirect daylight at laboratory temperature. Algae inoculum for the test was sampled from exponentially growing inoculum culture. The strain culture was always set to pre-culturing of cells for 3-4 days before the start of the test. Inoculum culture was kept 3-4 days under conditions at which the test was performed. After this period the culture was in the state of the exponential growth and had the cell density suitable for the performance of the test. The cell density of the pre-culture was measured just before the start of the test and the needed inoculum volume was calculated.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

21 - 24°C controlled at ± 2°C

pH:

7.6 - 7.8

Nominal and measured concentrations:

PRELIMINARY TESTNominal concentrations: 1 mg/L 0 h: 0.96 mg/L 72 h: 1.00 mg/LNominal concentrations: 100 mg/L 0 h: 99.4 mg/L 72 h: 101.7 mg/LNominal concentrations: 100 mg/L without algae 0 h: 100.2 mg/L 72 h: 104.0 mg/LLIMIT TESTNominal concentrations: 100 mg/L 0 h: 98.7 mg/L 72 h: 100.9 mg/LNominal concentrations: 100 mg/L without algae 0 h: 99.5 mg/L 72 h: 101.0 mg/L

Details on test conditions:

TEST SYSTEM - Test vessel: Erlenmeyers’ flasks - Aeration: without aeration - Initial cells density: 5000 cells per mL - Control end cells density: Burker´s counting chamber - No. of vessels per concentration (replicates): 3 in preliminary test, 6 in limit test - No. of vessels per control (replicates): 3 in preliminary test, 6 in limit test GROWTH MEDIUM - Standard medium used: yes OTHER TEST CONDITIONS - Adjustment of pH: no - Photoperiod: continuous white light - Light intensity and quality: 4 440 lux to 8 880 lux EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : - Determination of cell concentrations: Burker´s counting chamber - Observation intervals: 0, 24, 48 and 72 hours TEST CONCENTRATIONS - Range finding study - Test concentrations: 1, 5, 10 50 and 100 mg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate (CAS: 7778-50-9)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

see Table 1 and 2

Results with reference substance (positive control):

RESULTS OF REFERENCE TESTErC50 = 1.05 mg/L (potassium dichromate) (72 h)INTERLABORATORY TEST DATAFrom the results of the last interlaboratory test arranged by CSlab spol. s r.o. in 2016 we have obtained data for potassium dichromate 72h – ErC50 generated at 13 laboratories. The following reference range of values could be calculated from those data: ErC50 = 0.54 – 1.26 mg/L (72 h)

Table 1: Preliminary test results

Nominal concentration (mg/L)	Replicates	Cell density in 1 mL				Growth rate		Yield
		0 h	24 h	48 h	72 h	Mean	Reduction (%)	Mean	Inhibition (%)
100	1	5000	18750	37500	125000	1.07	13.4	117917	40.2
	2	5000	31250	50000	112500
	3	5000	25000	43750	131250
50	1	5000	25000	50000	131250	1.10	10.8	130417	33.8
	2	5000	25000	43750	143750
	3	5000	18750	56250	131250
10	1	5000	31250	50000	143750	1.11	9.6	136667	30.7
	2	5000	31250	50000	137500
	3	5000	25000	56250	143750
5	1	5000	31250	62500	150000	1.14	7.3	149167	24.3
	2	5000	25000	56250	143750
	3	5000	31250	62500	168750
1	1	5000	31250	62500	162500	1.16	5.5	159583	19.0
	2	5000	25000	62500	168750
	3	5000	31250	68750	162500
Control	1	5000	25000	87500	187500	1.23	-	197083	-
	2	5000	31250	75000	225000
	3	5000	25000	62500	193750

Table 2: Limit test results

Nominal concentration (mg/L)	Replicates	Cell density in 1 mL				Growth rate		Yield
		0 h	24 h	48 h	72 h	Mean	Reduction (%)	Mean	Inhibition (%)
100	1	5000	25000	43750	137500	1.10	7.2	130417	23.3
	2	5000	25000	50000	150000
	3	5000	18750	37500	118750
	4	5000	18750	56250	143750
	5	5000	31250	50000	125000
	6	5000	25000	56250	137500
Control	1	5000	18750	62500	137500	1.19	-	170000	-
	2	5000	25000	75000	175000
	3	5000	25000	56250	162500
	4	5000	25000	68750	162500
	5	5000	31250	87500	212500
	6	5000	25000	87500	200000

Validity criteria fulfilled:

yes

Conclusions:

The inhibition of growth rate was 7.2 % and the inhibition of yield was 23.3 % in the limit test. Therefore exact value of ErC50 and EyC50 could not be calculated and the values of EC are given in the form of a range.The determination of NOEC value was done by ANOVA (Analysis of Variance) analysis. Used ANOVA method is the part of statistical software QC.Expert 2.5 1998-2000 (product of TriloByte Ltd., Czech Republic). There is stated in the guideline that, if evidence is available to demonstrate that the concentration of the test substance has been satisfactorily maintained within ± 20 per cent of the nominal or measured initial concentration throughout the test, the results can be based on nominal or measured initial values. So the nominal concentrations were used for all evaluations and results.ErC50 > 100 mg/L (72 h, Desmodesmus subspicatus) (nominal concentration)EyC50 > 100 mg/L (72 h, Desmodesmus subspicatus) (nominal concentration)NOECr < 1 mg/L (72 h, Desmodesmus subspicatus) (nominal concentration)NOECy < 1 mg/L (72 h, Desmodesmus subspicatus) (nominal concentration)

Executive summary:

The test substance, Direct Yellow 28, was tested for growth inhibition on algae Desmodesmus subspicatus. The test was performed according to method C.3. - Freshwater Algae and Cyanobacteria, Growth Inhibition Test, Commission Regulation (EU) No.2016/266.

The preliminary test was performed in a range of the test substance nominal concentrations 1 – 100 mg/L. The preliminary test was performed twice. The first preliminary test was stopped due to the test would not meet the validity criteria.

The analytical results showed, that the test substance Direct Yellow 28 was sufficiently stable in the test medium at conditions of the test in the concentration range 1 – 100 mg/L. The highest inhibition of growth rate was 13.4 % and the highest inhibition of yield was 40.2 % in the preliminary test. Based on no toxicity of the test substance found in the preliminary test, the limit test was performed subsequently. The concentration of 100 mg/L was tested in the limit test. The inhibition of growth rate was 7.2 % and the inhibition of yield was 23.3 %.

The guideline specify that if evidence is available to demonstrate that the concentration of the test substance in the limit test has been satisfactorily maintained within ± 20 per cent of the nominal or measured initial concentration throughout the limit test, then the results can be based on nominal or measured initial values, which is the case of this study. The nominal concentrations were used for all evaluations and results.

ErC50 > 100 mg/L (72 h, Desmodesmus subspicatus) (nominal concentration)

EyC50 > 100 mg/L (72 h, Desmodesmus subspicatus) (nominal concentration)

NOECr < 1 mg/L (72 h, Desmodesmus subspicatus) (nominal concentration)      

NOECy < 1 mg/L (72 h, Desmodesmus subspicatus) (nominal concentration)

Description of key information

The effective concentration EC50 > 100 mg/L was determined based on inhibition of growth rate of aquatic algae.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L