Anthraquinone acid dye

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:

24 November 2008 and 16 January 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols with no deviations from standard test guidelines and/or minor methodological deficiences which do not affect the quality of the relevant results

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

OECD Guideline 201 further modified following OECD Guidance Document on Aquatic Toxicity Testing of Difficult Test Substances and Mixtures with regard to coloured test substances.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

EU Method C.3 further modified following OECD Guidance Document on Aquatic Toxicity Testing of Difficult Test Substances and Mixtures with regard to coloured test substances.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The test material concentration in the test samples was determined spectrophotometrically using an external standard. The method was developed by the Department of Analytical Services, Harlan Laboratories Ltd, Shardlow, UK. A volume of test sample was diluted with methanol to give a final theoretical concentration of 10 mg/l. Standard solutions of test material were prepared in methanol at a nominal concentration of 10 mg/l.

- Sample storage conditions before analysis:
Room temperature in the dark
Samples were taken from the control (replicates RI - R6 pooled) and the 100 mg/I test group (replicates RI - R3 and R4 - R6 pooled) at 0 and 72 hours for quantitative analysis. Duplicate samples were taken at 0 and 72 hours and stored at approximately -20°C for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- An amount of test material (100 mg) was dissolved in culture medium and the volume adjusted to 500 ml to give a 200 mg/I stock solution from which a series of dilutions was made to give further stock solutions of 20, 2.0 and 0.20 mg/I. An aliquot (25 ml) of each of the stock solutions was separately mixed with algal suspension (25 ml) to give the required test concentrations of 0.10, 1.0, 1 0 and 100 mg/I.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.


- Details of culture medium
NaNO3 25.5 mg/l
MgCI2.6H20 12.164 mg/l
CaCI2.2H20 4.41 mg/l
MgSO4.7H20 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15 mg/l
H3BO3 0.1855 mg/l
MnCI2.4H20 0.415 mg/l
ZnCI2 0.00327 mg/l
FeCI3.6H20 0.159 mg/l
CoCI2.6H20 0.00143 mg/l
Na2MoO4.2H20 0.00726 mg/l
CuCI2.2H20 0.000012 mg/I
Na2EDTA.2H20 0.30 mg/I
Na2SeO3.5H20 0.000010 mg/I

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: CCAP 276/20
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Sctoland
-Initial cell density approximately 10^3 cells/mL increased to 10^4 - 10^5 cells/mL
- Method of cultivation:Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1°C.

ACCLIMATION
- Culturing media and conditions (same as test or not): as test
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
Culture medium:
NaNO3 25.5 mg/l
MgCI2.6H2O 12.164 mg/l
CaCI2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3BO3 0.1855 mg/l
MnCI2.4H2O 0.415 mg/l
ZnCI2 0.00327 mg/l
FeCI3.6H2O 0.159 mg/l
CoCI2.6H2O 0.00143 mg/l
Na2MoO4.2H2O 0.00726 mg/l
CuCI2.2H2O 0.000012 mg/l
Na2EDTA.2H2O 0.30 mg/l
Na2SeO3.5H2O 0.000010 mg/l

The culture medium was prepared using reverse osmosis purified deionised water and the pH adjusted to 7.5 ± 0.1 with 0.1 N NaOH or HCI.
Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in
the laboratory under constant aeration and constant illumination at 21 ± 1°C.
- Any deformed or abnormal cells observed: All test and control cultures were inspected microscopically at 72 hours. There were no
abnormalities detected in any of the control or test cultures at 72 hours.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

nda

Test temperature:

21 ± 1°C

pH:

The culture medium was prepared using reverse osmosis purified deionised water* and the pH adjusted to 7.5 ± 0.1 with 0.1 N NaOH or HCI. The pH values of the control cultures (see Table 3) were observed to increase from pH 7.3 at 0 hours to pH 7.7 - 7.8 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines

Dissolved oxygen:

nda

Salinity:

nda

Nominal and measured concentrations:

Concentrations used in the range-finding test
- 0.10, 1.0, 1 0 and 100 mg/I
Concentration used in the definitive test
- Based on the result of the range-finding test a "limit test" was conducted at a concentration of 100 mg/I to confirm that at the maximum concentration given in the OECD/EEC Test Guidelines no effect on algal growth was observed.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 94% to 109% of nominal and so it was considered justifiable to estimate the EC50 values in terms of the nominal test concentrations only.

Details on test conditions:

TEST SYSTEM
- Test vessel:
The test was conducted in 250 ml glass conical flasks each containing 25 ml of test preparation and plugged with polyurethane foam bungs to
reduce evaporation. Two replicate flasks were used for each control and test concentration. The test material was dissolved directly in culture
medium.
- Initial cells density:
Prior to the start of the test sufficient master culture was added to approximately 100 ml volumes of culture media contained in conical flasks
to give an initial cell density of approximately 103 cells/ml. The flasks were plugged with polyurethane foam stoppers and kept under constant
agitation by orbital shaker (100 - 150 rpm) and constant illumination at 24 ± 1°C until the algal cell density was approximately 104 - 105
cells/ml.
- Control end cells density:
Mean cell density of control at 0 hours: 5.32 x 10^3 cells per ml
Mean cell density of control at 72 hours: 1.05 x 10^5 cells per ml

- No. of organisms per vessel: n/a
- No. of vessels per concentration (replicates): 6 flasks
- No. of vessels per control (replicates): 6 flasks
- At initiation of the test, the culture contained a nominal cell density of 4 x 10^3 cells/mL
GROWTH MEDIUM
Culture medium:
NaNO3 25.5 mg/l
MgCI2.6H2O 12.164 mg/l
CaCI2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3BO3 0.1855 mg/l
MnCI2.4H2O 0.415 mg/l
ZnCI2 0.00327 mg/l
FeCI3.6H2O 0.159 mg/l
CoCI2.6H2O 0.00143 mg/l
Na2MoO4.2H2O 0.00726 mg/l
CuCI2.2H2O 0.000012 mg/l
Na2EDTA.2H2O 0.30 mg/l
Na2SeO3.5H2O 0.000010 mg/l

The culture medium was prepared using reverse osmosis purified deionised water and the pH adjusted to 7.5 ± 0.1 with 0.1 N NaOH or HCI.

OTHER TEST CONDITIONS
- Sterile test conditions: nda
- Adjustment of pH:
The culture medium was prepared using reverse osmosis purified deionised water and the pH adjusted to 7.5 ± 0.1 with 0.1 N NaOH or HCI.
- Photoperiod: Constant Illumination
- Light intensity and quality: Approximately 10000 lux) provided by warm white lighting (380 - 730 nm)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations:
Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a
Coulter® Multisizer Particle Counter.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations:
An amount of test material (100 mg) was dissolved in culture medium and the volume adjusted to 500 ml to give a 200 mg/l stock solution
from which a series of dilutions was made to give further stock solutions of 20, 2.0 and 0.20 mg/l. An aliquot (25 ml) of each of the stock
solutions was separately mixed with algal suspension (25 ml) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/l.
The range-finding test was conducted by exposing Desmodesmus subspicatus cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/l for a period of 72 hours.
- Results used to determine the conditions for the definitive study:
Based on the result of the range-finding test a "limit test" was conducted at a concentration of 100 mg/l to confirm that at the maximum concentration given in the OECD/EEC Test Guidelines no effect on algal growth was observed.

Reference substance (positive control):

yes

Remarks:

potassion dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures at 72 hours.

At both the start and end of the test the control cultures were observed to be clear colourless solutions whilst the 100 mg/I test cultures were observed to be red solutions.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50:

A positive control (Harlan Laboratories Ltd Project No: 0039/1066) used potassium dichromate as the reference material at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/I.

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of Desmodesmus subspicatus (CCAP 276/20) to the reference material gave the following results:
ErC50 (0 - 72 h) : 0.52 mg/I, 95% confidence limits 0.43 - 0.62 mg/I
EyC50 (0 - 72 h) : 0.29 mg/I, 95% confidence limits 0.25 - 0.33 mg/I
EbC50 (0 - 72 h) : 0.30 mg/I, 95% confidence limits 0.26 - 0.34 mg/I

No Observed Effect Concentration (NOEC) based on growth rate: 0.125 mg/I
No Observed Effect Concentration (NOEC) based on yield: 0.125 mg/I
No Observed Effect Concentration (NOEC) based on biomass integral: 0.0625 mg/I

Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.25 mg/I
Lowest Observed Effect Concentration (LOEC) based on yield: 0.25 mg/I
Lowest Observed Effect Concentration (LOEC) based on biomass integral:0.125 mg/I

The results from the positive control with potassium dichromate were within the normal ranges for this reference material.

Any other information on results incl. tables

Table 1. Cell densities and percentage inhibition of growth from the range-finding test

	Cell Density (cells/mL)	Cell Density (cells/mL)	Inhibition Values (%)	Inhibition Values (%)
Nominal Concentration (mg/L)	0 Hours	72 Hours	Growth Rate*	Yield/Biomass Integral*
Control Replicate 1	4.06E+03	2.62E+05
Control Replicate 2	4.17E+03	1.41E+05
Control Mean	4.11E+03	2.01E+05
0.10 Replicate 1	4.20E+03	2.33E+05	4	21
0.10 Replicate 2	4.21E+03	2.52E+05	4	21
0.10 Mean	4.20E+03	2.43E+05	4	21
1.0 Replicate 1	4.35E+03	2.77E+05	7	35
1.0 Replicate 2	4.15E+03	2.63E+05	7	35
1.0 Mean	4.25E+03	2.70E+05	7	35
10 Replicate 1	4.08E+03	2.48E+05	4	19
10 Replicate 2	4.34E+03	2.30E+05	4	19
10 Mean	4.21E+03	2.39E+05	4	19
100 Replicate 1	4.16E+03	2.79E+05	9	52
100 Replicate 2	4.35E+03	3.30E+05	9	52
100 Mean	4.26E+03	3.05E+05	9	52
*Increase in growth compared to controls

Table 5. Inhibition of growth rate, yield, and biomass integral in the definitive test

	Growth Rage (cells/mL/hour)	Growth Rage (cells/mL/hour)	Yield (cells/mL)	Yield (cells/mL)	Biomass integral (cells/mL/hour)	Biomass integral (cells/mL/hour)
Nominal Concentration (mg/L)	0 - 72 h	% Inhibition*	0 - 72 h	% Inhibition*	0 - 72 h	% Inhibition*
Control Rep 1	0.041		6.87E+04		1.80E+06
Control Rep 2	0.046		1.04E+05		2.11E+06
Control Rep 3	0.044		9.08E+04		1.67E+06
Control Rep 4	0.048		1.26E+05		2.52E+06
Control Rep 5	0.044		9.32E+04		2.00E+06
Control Rep 6	0.047		1.16E+05		2.06E+06
Control Mean	0.045		9.97E+04		3.11E+05
Control SD	0.003		2.01E+04		2.29E+06
100 mg/L Rep 1	0.044	-2	9.15E+04		2.29E+06	11
100 mg/L Rep 2	0.05	11	1.43E+05		2.45E+06	19
100 mg/L Rep 3	0.051	13	1.50E+05		3.33E+06	61
100 mg/L Rep 4	0.06	33	2.94E+05		5.45E+06	164
100 mg/L Rep 5	0.057	27	2.45E+05		4.54E+06	120
100 mg/L Rep 6	0.052	16	1.66E+05		3.65E+06	77
100 mg/L Mean	0.052	16	1.81E+05	82	3.62E+06	75
100 mg/L SD	0.006		7.42E+04		1.22E+06
*Positive values indicate increase in growth as compared to controls

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See overall remarks

Conclusions:

The effect of the test material on the growth of Desmodesmus subspicatus has been investigated and gave EC50 values of greater than 100
mg/l. Correspondingly the No Observed Effect Concentration was 100 mg/l.

Executive summary:

In an algal growth inhibition test on the growth of Desmodesmus subspicatus (Harlan project number: 0959/0242) the

test material was found to have EC50values of greater than 100 mg/l. Correspondingly the No Observed Effect

Concentration was 100 mg/l.

The method followed the recommendations of the OECD Guidelines for Testing of Chemicals (2006) No 201,

"Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation

(EC) No. 440/2008 and further modified following the OECD Guidance Document on Aquatic Toxicity Testing of

Difficult Test Substances and Mixtures with regards to coloured test substances.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/. Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 94% to 109% of nominal and so the results are based on nominal test concentrations only.