Chlorinated reaction product of Vat Blue 20 (Violanthrone A, Violanthrone B and Iso-violanthrone) - Earlier known as CI Vat Blue 18

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:

Study Initiation date : September 13, 2017 and Study Completion : February 13, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Storage Temperature : Room temperature (28 - 32 °C)

Vehicle:

no

Remarks:

Based on the solubility, algal medium of the test was selected as the vehicle for study.

Details on test solutions:

A quantity of 100 mg test item was transferred in 1000 mL beaker and volume was made up to 800 mL with algal medium to obtain the nominal concentration of 0.125 mg/mL (stock A). Stock A was sonicated for at least 2 h and then filtered. After filtration, stock solution was found light blue in colour.
Volumes of 1.248, 2.4, 4.8, 9.6 and 19.2 mL from the stock A and 1000 μL algal culture were taken and diluted to 120 mL with sterile algal culture medium in respective beakers and after sampling transferred to conical flasks of 250 mL capacity to obtain the nominal test concentrations of 1.3, 2.5, 5.0, 10.0 and 20.0 mg test item/L respectively.
For control group, only 1000 μL alga culture was transferred using a micropipette and diluted to 120 mL with sterile algal culture medium
Six replicates for the control group and three replicates for the treatment groups were used.
The experiment was conducted in 250 mL conical flasks.

Test organisms (species):

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

Details on test organisms:

Name of the Organism : Pseudokirchneriella subcapitata
Strain Number : ATCC 22662
Origin and Source of Culture : American Type Culture Collection, 10801, University of Boulevard, Manassas, Virginia, 20110-2209, USA
Subculture Maintained at : Section - Ecotoxicology Jai Research Foundation Valvada, Dist. Valsad, Gujarat
Date of Receipt (at JRF) : March 10, 2017
Date of Last Subculture : December 04, 2017
Method of Cultivation : Static condition

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

no

Hardness:

Not specified

Test temperature:

21 - 23 °C

pH:

7.70– 7.96

Dissolved oxygen:

not measured

Salinity:

as of freshwater conditions

Conductivity:

not measured

Nominal and measured concentrations:

0.0 (control), 1.3, 2.5, 5.0, 10.0 and 20.0 mg test item/L

Details on test conditions:

TEST SYSTEM
Temperature (°C) : 21 - 23 °C (APPENDIX 2)
Test Duration : 72 h
Mean Illumination (lux) : 6250-6747 (± 15% of the mean value)
pH Values Range : 7.70– 7.96
Culturing Apparatus : 250 mL sterile conical flask
Test Concentrations : 0.0 (control), 1.3, 2.5, 5.0, 10.0 and 20.0 mg test item/L
Initial Mean Number of (Cells/mL) : 6917
Six replicates for the control group and three replicates for the treatment groups were used.
The experiment was conducted in 250 mL conical flasks.
GROWTH MEDIUM
- Standard medium used: yes/no
- Detailed composition if non-standard medium was used:

TEST MEDIUM / WATER PARAMETERS
Algal Culture Medium
Stock Solution Preparation for Culture Media
-preparation of dilution water:
-Stock Solution I : Macro Nutrients
Ammonium Chloride Pure (NH4Cl) : 1.5 g/L
Magnesium Chloride Hexahydrate (MgCl2.6H2O): 1.2 g/L
Calcium Chloride Dihydrate (CaCl2.2H2O): 1.8 g/L
Magnesium Sulphate Heptahydrate (MgSO4.7H2O): 1.5 g/L
Potassium Dihydrogen Phosphate Purified (KH2PO4): 0.16 g/L
-Stock Solution II : Iron
Ferric Chloride Hexahydrate (FeCl3.6H2O): 64 mg/L
EDTA Disodium Salt (C10H14N2O8Na2·2H2O): 100 mg/L
-Stock Solution III : Trace elements
Boric Acid (H3BO3): 185 mg/L
Manganese (II) Chloride tetrahydrate (MnCl2.4H2O): 415 mg/L
Zinc Chloride (ZnCl2): 3 mg/L
Cobalt Chloride Hexahydrate (CoCl2.6H2O): 1.5 mg/L
Copper (II) Chloride dihydrate (CuCl2.2H2O): 0.01 mg/L
Sodium Molybdate, dihydrate (Na2MoO4.2H2O): 7 mg/L
-Stock Solution IV : Bicarbonate
Sodium Hydrogen Carbonate (NaHCO3): 50 g/L
Stock solution I and III were sterilized by autoclaving (120 °C for 15 min), Stock solution II and IV were filtered through membrane (0.2 μm) and refrigerated.
-Algal Culture Medium Preparation: Culture medium was prepared by adding stock solution I (10 mL), stock solution II (1 mL), stock solution III (1 mL) and stock solution IV (1 mL) and equated up to 1 L in sterilized deionised water.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH:
- Photoperiod: 24 h
- Light intensity and quality: 6717-6770 lux (± 15% of mean value) with a universal UV- free white fluorescent lamp.
The cells in the cultured flasks were maintained in suspension by agitating the test flasks continuously at 100 +/- 2 rpm, using an orbital shaker

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations:
- Chlorophyll measurement:


TEST CONCENTRATIONS
-0.0 (control), 1.3, 2.5, 5.0, 10.0 and 20.0 mg test item/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

2.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Validity criteria fulfilled:

yes

Conclusions:

Due to insolubility of test item in water and vehicles (Acetone, DMSO and DMF) analytical method cannot be established. Hence, the study was conducted with filtrate after mixing of test item in test media and results are expressed based on the nominal concentration of test item. The colour of the test solution was light blue. The light blue coloured liquid can absorb light and hence, limit the growth of algal cultures. Absorption is proportional to intensity of colour and as a consequence it may have result in growth inhibition.
Please find below ECx values for growth rate inhibition, biomass inhibition and yield.
EbC10 for biomass inhibition (0 - 72 h) : 1.98 mg/L
ErC10 for growth rate inhibition (0 - 72 h) : 2.53 mg/L
EyC10 for yield inhibition (0 - 72 h) : 2.24 mg/L
EbC20 for biomass inhibition (0 - 72 h) : 2.43 mg/L
ErC20 for growth rate inhibition (0 - 72 h) : 3.62 mg/L
EyC20 for yield inhibition (0 - 72 h) : 2.83 mg/L
EbC50 for biomass inhibition (0 - 72 h) : 3.59 mg/L
ErC50 for growth rate inhibition (0 - 72 h) : 7.19 mg/L
EyC50 for yield inhibition (0 - 72 h) : 4.43 mg/L
NOEC for biomass, growth rate and yield : 1.3 mg/L
LOEC for biomass, growth rate and yield : 2.5 mg/L

Executive summary:

This study was performed to assess the effects of the test substance on the growth of the alga Pseudokirchneriella subcapitata under static condition.

Exponentially growing cultures of P.subcapitata were exposed to nominal test concentrations of 0.0 (control), 1.3, 2.5, 5.0, 10.0 and 20.0 mg test item/L. The algal cultures were assessed for the growth by visual cell count at 24, 48 and 72 h.

A concentration-effect relationship was observed and statistically analysed to obtain effect concentrations.

The growth rate inhibition effect concentrations with their respective 95% lower and upper confidence limits for the inhibition of growth rate were ErC10 = 2.53 (1.57 – 4.07) mg/L, ErC20 = 3.62 (2.50 – 5.23) mg/L and ErC50 = 7.19 (5.53 – 9.36) mg/L

The biomass inhibition effect concentrations with their respective 95% lower and upper confidence limits for the biomass inhibition were EbC10 = 1.98 (1.76 – 2.22) mg/L, EbC20 = 2.43 (2.21 – 2.67) mg/L and EbC50= 3.59 (3.30 – 3.91) mg/L.

The yield inhibition effect concentrations with their respective 95% lower and upper confidence limits for the yield were EyC10 = 2.24 (1.75 – 2.87) mg/L, EyC20 = 2.83 (2.29 – 3.50) mg/L and EyC50 = 4.43 (3.58 –5.47) mg/L.

Based on the results of the statistical analysis, the NOEC and LOEC values for biomass (cell growth), growth rate and yield were 1.3 and 2.5 mg test item/L, respectively.

Description of key information

This study was performed to assess the effects of the test substance on the growth of the alga Pseudokirchneriella subcapitata under static condition.

Exponentially growing cultures of P.subcapitata were exposed to nominal test concentrations of 0.0 (control), 1.3, 2.5, 5.0, 10.0 and 20.0 mg test item/L. The algal cultures were assessed for the growth by visual cell count at 24, 48 and 72 h.

A concentration-effect relationship was observed and statistically analysed to obtain effect concentrations.

The growth rate inhibition effect concentrations with their respective 95% lower and upper confidence limits for the inhibition of growth rate were ErC10 = 2.53 (1.57 – 4.07) mg/L, ErC20 = 3.62 (2.50 – 5.23) mg/L and ErC50 = 7.19 (5.53 – 9.36) mg/L

The biomass inhibition effect concentrations with their respective 95% lower and upper confidence limits for the biomass inhibition were EbC10 = 1.98 (1.76 – 2.22) mg/L, EbC20 = 2.43 (2.21 – 2.67) mg/L and EbC50= 3.59 (3.30 – 3.91) mg/L.

The yield inhibition effect concentrations with their respective 95% lower and upper confidence limits for the yield were EyC10 = 2.24 (1.75 – 2.87) mg/L, EyC20 = 2.83 (2.29 – 3.50) mg/L and EyC50 = 4.43 (3.58 –5.47) mg/L.

Based on the results of the statistical analysis, the NOEC and LOEC values for biomass (cell growth), growth rate and yield were 1.3 and 2.5 mg test item/L, respectively.

Additional information

NOEC and LOEC values for biomass (cell growth), growth rate and yield were 1.3 and 2.5 mg test item/L, respectively.