Lithium 4-[(5-amino-3-methyl-1-phenyl-1H-pyrazol-4-yl)azo]-2,5-dichlorobenzenesulphonate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Justification for type of information:

Justification for Read Across is reported in the endpoint summary and it is further detailed in the report attached to the IUCLID section 13.

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 (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis.
All 0-hour samples were stored frozen prior to analysis.
Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours.

Vehicle:

no

Details on test solutions:

A nominal amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1 liter to give a 100 mg/l stock solution from which a series of dilutions was made to give further stock solutions of 32, 10, 3.2 and 1.0 mg/l. An aliquot (450 ml) of each of the stock solutions was separately inoculated with algal suspension (5.3 ml) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/l.

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

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name:
- Strain: strain CCAP 278/4
- Source: liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Age of inoculum (at test initiation):
- 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.
- Preparation for inoculum: 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 1E+03 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 1E+04 - 1E+05 cells/ml.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

24 ± 1 °C

pH:

Control: 7.2 - 8.2
Test vessels: 7.3 - 8.2

Nominal and measured concentrations:

Nominal: 1.0, 3.2, 10, 32 and 100 mg/l.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml glass conical flasks.
- Material, size, headspace, fill volume: 25 ml of test preparation.
- Initial cells density: pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 4.23 x 10^5 cells per ml. Inoculation of 450 ml of test medium with 5.3 ml of this algal suspension gave an initial nominal cell density of 5 x 10^3 cells per ml and had no significant dilution effect on the final test concentration.
- No. of vessels per concentration: 3 replicates.
- No. of vessels per control: 6 replicates.
- Incubation: the flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 °C.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline.

OTHER TEST CONDITIONS
- Photoperiod: under continuous illumination.
- Light intensity and quality: intensity approximately 10000 lux, provided by warm white lighting (380 – 730 nm).
- Stirring: constant shaken at approximately 150 rpm for 72 hours.

EFFECT PARAMETERS MEASURED
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.
To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerized interpolation using the Xlfit software package (IDBS). ECxvalues were then determined from the equation for the fitted line.

TEST CONCENTRATIONS - RANGE FINDING
- Range finding study: the results of the initial range-finding test showed inhibition of growth occurred, particularly at 100 mg/l where significant light absorption was observed and so a second range-finding test was conducted in accordance with the recommendations of the OECD Guidance Document No. 23 on Aquatic Testing of Difficult Substances and Mixtures for the testing of colored substances.
- Concentration: nominal test concentrations of 0.10, 1.0, 10 and 100 mg/l

VALIDITY CRITERIA
The results of the test are considered valid if following performance criteria are met:
- The cell concentration of the control cultures must increased by a factor of at least 16 over the test period.
- The mean coefficient of variation for section-by-section daily growth rates in the control cultures during the course of the test (days 0-1, 1-2 and 2-3, for 72-hour tests) must not exceed 35 %.
- The coefficient of variation of average specific growth rate in replicate control cultures must not exceed 7 %.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Details on results:

Growth rate (r) and yield (y) of Pseudokirchneriella subcapitata were not significantly affected by the presence of the test item over the 72-Hour exposure period. Accordingly the following results were determined from the data:

ErC10 (0 - 72 h) > 100 mg/l (nominlal)
LOErC (0 - 72 h): 100 mg/l (nominlal)
EyC10 (0 - 72 h): 28 mg/l (nominlal)
LOEyC (0 - 72 h): 100 mg/l (nominlal)

There were no statistically significant decreases in growth rate, between the control, 1.0, 3.2, 10 and 32 mg/l test concentrations however the 100 mg/l test concentration was significantly different and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 32 mg/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 100 mg/l.

There were no statistically significant decreases in yield, between the control, 1.0, 3.2, 10 and 32 mg/l test concentrations however the 100 mg/l test concentration was significantly different and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 32 mg/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 100 mg/l.

TEST CONCENTRATIONS
Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 88 % to 106 % of nominal and so the results are based on nominal test concentrations only.

RANGE-FINDING TEST
The results of the range-finding tests showed no effect on growth rate at the test concentrations of 0.10, 1.0 and 10 mg/l. However, growth rate was observed to be reduced at 100 mg/l.
Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg/l were selected for the definitive test.

Chemical analysis of the 1.0, 10 and 100 mg/l test preparations taken from the initial range-finding test at 0 and 72 hours showed measured concentrations to range from 87 % to 95 % of nominal indicating that the test item was stable under test conditions.

Results with reference substance (positive control):

A positive control used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/l.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 – 72 h): 1.2 mg/L; 95% confidence limits 1.1 – 1.4 mg/l
EyC50 (0 – 72 h): 0.63 mg/L; 95% confidence limits 0.57 – 0.70 mg/l

No Observed Effect Concentration (NOEC) based on growth rate: 0.50 mg/l
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate: 1.0 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/l

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

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the growth rate and yield data after 72 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Validity criteria fulfilled:

yes

Conclusions:

ErC50 (72h) > 100 mg/l (nominal)
EyC50 (72h) > 100 mg/l (nominal)

Executive summary:

Method

The substance was tested for growth inhibition on algae Pseudokirchneriella subcapitata, according to the OECD guideline 201. Following preliminary range-finding tests, algae was exposed to an aqueous solution of the test item at the concentration of 1.0, 3.2, 10, 32 and 100 mg/l (three replicates per concentration), over a period of 72 hours.

Growth rate (r) and yield (y) of Pseudokirchneriella subcapitata were not significantly affected by the presence of the test item over the 72-Hour exposure period.

There were no statistically significant decreases in growth rate and/or yield, between the control, 1.0, 3.2, 10 and 32 mg/l test concentrations however the 100 mg/l test concentration was significantly different and, therefore the "No Observed Effect Concentration" (NOEC) in both cases of growth rate and yield was 32 mg/l; correspondingly the "Lowest Observed Effect Concentration" (LOEC) was 100 mg/l, in both cases of growth rate and yield.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 88 % to 106 % of nominal and so the results are based on nominal test concentrations only.

Results

ErC50 (72h) > 100 mg/l (nominal)

EyC50 (72h) > 100 mg/l (nominal)

Description of key information

Not harmful/toxic to aquatic algae and cyanobacteria