Reaction products of 1,3-dihydroxybenzene, 4-amino-5-hydroxynaphthalene-2,7-disulphonic acid, 4-methoxy-1-aminobenzene, 2-(4-aminoanilino)-5-nitrobenzenesulphonic acid, sodium salt

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:

2016

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) 2016/266

Deviations:

yes

Remarks:

see Any other information about materials and methods

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Test substance name: Acid Brown 235
- Other name: Korostan Brown DGR
- Source and lot/batch No. of test material: 7012/2007
- Expiration date of the lot/batch: May 2018
- Appearance: dark brown powder

Analytical monitoring:

yes

Details on sampling:

PRELIMINARY TEST
The 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 (10 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 analysed 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 analysed on the day of delivery. All samples were stored at laboratory temperature. The analytical results showed that the test substance was stable in test medium under the conditions of the test.

FULL TEST
The 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 (120 mg/L), the lowest (10 mg/L) test concentration and from concentration (200 mg/L) without algae. The samples for analysis were taken at the beginning of the test (0 hours) and immediately delivered in transport box to analytical laboratory. 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 analysed on the day of delivery. All samples were stored at laboratory temperature.

Vehicle:

no

Details on test solutions:

BASIC STOCK SOLUTIONS
The 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 mL
Solution 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 mL

PREPARATION OF THE TEST MEDIUM
The 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.

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): The Botanic Institute of the Czech Academy of Science, Třeboň on date 27.5.2016

ACCLIMATION
The strain culture was preinoculated from the stock culture 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. The algal cultures did not contain deformed or abnormal cells.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21.5 – 22.0°C (preliminary test)
22.0 – 22.5°C (full test)

pH:

7.6 (preliminary test)
7.7 (full test)

Nominal and measured concentrations:

Nominal concentration: 10 mg/L
8.59 mg/L (0 h)
8.26 mg/L (72 h)
Nominal concentration: 120 mg/L
110.8 mg/L (0 h)
116.9 mg/L (72 h)
Nominal concentration: 200 mg/L (without algae)
187.4 mg/L (0 h)
196.1 mg/L (72 h)

Details on test conditions:

INSTRUMENTS AND EQUIPMENT
­ test area temperature control equipment
­ ramp with white fluorescent lamp
­ fluorescent lamps type: Philips TLD 36W/33, white light, wattage: 36 Watt
­ microscope
­ Erlenmeyer flasks with stoppers venting air
­ Burker´s counting chamber
­ sterilizer
­ analytical balance AG 135 (Mettler Toledo)
­ laboratory shaker LT2
­ pH metr WTW inoLab pH 730
­ conductivity meter FE 30 (Mettler Toledo)
­ ultrasound Bandelin Sonorex
­ volumetric flasks, pipettes and another common laboratory equipment

TEST CONDITIONS
- Temperature: 21 – 24°C controlled at ± 2°C
- Lighting: under continuous white light, 4 440 lux to 8 880 lux
- Exposition time: 72 hours
- Volume of tested mixture: 20 mL
- Initial concentration of algae culture: 5 000 cells in a 1 mL
- Aeration: without aeration.
- Agitation of algae culture by shaking.

PREPARATION OF SOLUTIONS OF THE TEST SUBSTANCE
The stock solution of the test substance was prepared in the test medium. 100 mg of the test substance was weighed into 1000 mL of the test medium for the preliminary test and 100 mg of the test substance was weighted into 500 mL of the test medium for the full test. The stock solutions were ultrasonicated for 5 minutes in the full test.

PRELIMINARY TEST
The stock solution of the test substance in test medium was prepared for the preliminary test. The test was performed in a range nominal concentrations from 10 mg/L to 100 mg/L. Testing mixtures were prepared by dosing appropriate volumes of the stock solution of the test substance and inoculum into volumetric flasks. Testing mixtures were incubated in Erlenmeyers’ flasks. The test contained three parallel series of the test substance concentrations and three controls without the test substance. The volume of the test solution was 20 mL in each testing flask. The initial density of 5 000 cells per mL was used in the test. The flasks were placed on a shaker under the lighting ramp and were incubated under continuous illumination and shaking for 72 hours. At the beginning and the end of the test the pH of the test mixtures was measured. The light intensity and temperature were measured every 24 hours. The density of algae culture was evaluated microscopically at 24, 48 and 72 hours. The cell density was measured by direct counting of living cells in Burker´s counting chamber. The growth rates (µ) and the yield (Y) and percentage reduction of growth rate and percentage inhibition of yield were calculated from obtained values.
- Stock solution of the test substance: 100.03 mg/L
- Test concentrations: 100, 80, 40, 20 and 10 mg/L
- Conductivity of deionized water: 2.26 µS/cm
- pH of the test medium: 7.6
- Volume of inoculated algae culture: 3.2 mL in 200 mL of mixture
- Lighting during the test: 8345 – 8365 lux
- Temperature during the test: 21.5 – 22.0°C

FULL TEST
The full test was performed in a range of the test substance nominal concentrations 10 – 120 mg/L. The stock solution of the test substance in the test medium was prepared for the full test. The test was performed in range of nominal concentrations from 10 mg/L to 100 mg/L. For the test the geometric concentration series of the test substance was used with factor 1.5. Testing mixtures were prepared by dosing appropriate volumes of stock solution of the test substance and inoculum into volumetric flasks. Testing mixtures were incubated in Erlenmeyers’ flasks. The test contained three parallel series of the test substance concentrations and three controls without the test substance. The volume of the test solution was 20 mL in each testing flask. The initial density of 5 000 cells per mL was used in the test. The flasks were placed on a shaker under the lighting ramp and were incubated under continuous illumination and shaking for 72 hours. At the beginning and the end of the test the pH of the test mixtures was measured. The light intensity and temperature were measured every 24 hours. The density of algae culture was evaluated microscopically at 24, 48 and 72 hours. The cell density was measured by direct counting of living cells in Burker’s counting chamber. The growth rates (µ) and the yield (Y) and percentage reduction of growth rate and percentage inhibition of yield were calculated from obtained values.
- Stock solution of the test substance: 100.07 mg/500 mL
- Test concentration: 120, 80, 53, 35, 23, 15 and 10 mg/L
- Conductivity of deionized water: 1.30 µS/cm
- pH of test medium: 7.7
- Volume of inoculum algae culture: 0.62 mL in 200 mL mixture
- Lighting during the test: 8 345 – 8365 lux
- Temperature during the test: 22.0 – 22.5°C

DETERMINATION OF THE TEST SUBSTANCE CONCENTRATIONS
The determination of the test substance concentrations was carried out by HPLC method.
Preliminary test
The 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 (10 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 analysed 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 analysed on the day of delivery. All samples were stored at laboratory temperature.
Full test
The 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 (120 mg/L), the lowest (10 mg/L) test concentration and from concentration (200 mg/L) without algae. The samples for analysis were taken at the beginning of the test (0 hours) and immediately delivered in transport box to analytical laboratory. 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 analysed on the day of delivery. All samples were stored at laboratory temperature.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (CAS: 7778-50-9)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

124.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

22.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

9.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

see Table 1

Results with reference substance (positive control):

Measured value: ErC50 = 1.05 mg/L (72 h)
Interlaboratory test data: ErC50 = 0.54 - 1.26 mg/L (72 h)

Reported statistics and error estimates:

The value of ErC50, EyC50, ErC10, EyC10 and the confidence intervals were calculated using the statistical software ToxRat Professional Version 3.2.1.
The determination of the NOEC values were done by ANOVA (Analysis of Variance) analysis. Used ANOVA method is the part of the statistical software ToxRat Professional Version 3.2.1. The significant difference to control is 0.05.

Table 1: Growth rate

Nominal concentration (mg/L)	Mean cell density in 1 mL				Mean growth rate	Growth rate reduction (%)
	0 hours	24 hours	48 hours	72 hours	0.59	48.4
120	5000	6250	20833	29167	0.67	41.0
80	5000	12500	27083	37500	0.75	33.8
53	5000	16667	33333	47917	0.84	26.0
35	5000	18750	31250	62500	0.89	21.5
23	5000	20833	33333	72917	0.95	16.2
15	5000	18750	39583	87500	1.06	7.2
10	5000	22917	41667	118750	1.14	0.0
Control	5000	20833	50000	152083	0.59	48.4

Validity criteria fulfilled:

yes

Conclusions:

The analytical results showed, that the test substance, Acid Brown 235, was stable in the test medium at conditions of the test. Therefore the nominal concentrations were used for all evaluations and results.
Test results:
ErC50 = 124.1 mg/L (Desmodesmus subspicatus, 72 h) (nominal concentration)
EyC50 = 22.4 mg/L (Desmodesmus subspicatus, 72 h) (nominal concentration)

ErC10 = 9.1 mg/L (Desmodesmus subspicatus, 72 h) (nominal concentration)
EyC10 = 3.1 mg/L (Desmodesmus subspicatus, 72 h) (nominal concentration)

NOECr < 10 mg/L (Desmodesmus subspicatus, 72 h) (nominal concentration)
NOECy < 10 mg/L (Desmodesmus subspicatus, 72 h) (nominal concentration)

Executive summary:

The test substance, Acid Brown 235, was tested for growth inhibition on algae Desmodesmus subspicatus. The test was performed according method C.3. Freshwater Algae and Cyanobacteria, Growth Inhibition Test, Commission Regulation (EU) 2016/266.

The preliminary test was performed in a range of the test substance nominal concentrations 10 – 100 mg/L. Based on the toxicity 49.5 % (a value very close to 50 percent) of the test substance for the growth rate found in the preliminary test, the full test was performed in appropriate concentration range.

The analytical results showed, that the test substance Acid Brown 235 was stable in the test medium at conditions of the test in concentration range 10 – 100 mg/L.

The full test was performed in a range of the test substance nominal concentrations 10 – 120 mg/L.

The guideline specifies that if evidence is available to demonstrate that the concentration of the test substance in full test has been satisfactorily maintained within ± 20 per cent of the nominal or measured initial concentration throughout the full test, then the results can be based on nominal or measured initial values.

The nominal concentrations were used for all evaluations and results.

Test results:

ErC50 = 124.1 mg/L (Desmodesmus subspicatus, 72 h)(nominal concentration)

EyC50 = 22.4 mg/L (Desmodesmus subspicatus, 72 h)(nominal concentration)

 

ErC10 = 9.1 mg/L (Desmodesmus subspicatus, 72 h)(nominal concentration)

EyC10 = 3.1 mg/L (Desmodesmus subspicatus, 72 h)(nominal concentration)

 

NOECr < 10 mg/L (Desmodesmus subspicatus, 72 h) (nominal concentration)

NOECy < 10 mg/L (Desmodesmus subspicatus, 72 h) (nominal concentration)

Key value for chemical safety assessment

EC50 for freshwater algae:

124.1 mg/L

EC10 or NOEC for freshwater algae:

9.1 mg/L