4-hydroxycoumarin

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:

Experiment start date - 09 October 2020; Experiment end date - 02 December 2020.

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

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: 4-Hydroxycoumarin
CAS Number: 1076-38-6
Batch: HCN20002
Purity: >98%
Physical State/Appearance: Beige powder
Expiry Date: 26 September 2022
Storage Conditions: Room temperature in the dark

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 samples were analyzed on the day of sampling. Duplicate samples were taken at each occasion and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

A nominal amount of test item (1100 mg) was dispersed in 11 L of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Gelman Acrocap filter (first approximate 100 mL used to pre-condition the filter was discarded) to give a 100% v/v saturated solution.
A series of dilutions was made from this saturated solution to give stock solutions of 10, 18, 32 and 56% v/v saturated solution. An aliquot (500 mL) of each of the stock solutions was separately inoculated with 2.0 mL of algal suspension to give an initial nominal cell density of 5.00 x 103 cells/mL.
The stock solutions and each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

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

Details on test organisms:

The test was carried out using Raphidocelis subcapitata strain CCAP 278/4. Liquid cultures of Raphidocelis subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
Approximately 3 to 4 days before the start of the test, inoculum cultures of algae were set up at an initial cell density of approximately 1000 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 10E5 to 10E6 cells/mL.
A positive control test using potassium dichromate as the reference item was performed twice in a 12 month period to demonstrate satisfactory conditions of the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24 ±1 ºC

pH:

4.3 - 7.7

Nominal and measured concentrations:

Range finding test - 0 (Control), 0.1, 1, 10, 100 mg/L
Definitive test- 0 (control), 10, 18, 32, 56, 100 mg/L.

Details on test conditions:

As in the range-finding test, 250 mL glass conical flasks were used. Six flasks each containing 100 mL of solution were used for the control group and three flasks each containing 100 mL were used for each treatment group.
The control group was maintained under identical conditions but not exposed to the test item.
Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 1.27 x 10E6 cells per mL. Inoculation of 500 mL of test medium with 2.0 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10E3 cells per mL and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron incubator) at 24 ±1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

47 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

40 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Range-finding Test
The results showed no effect on growth at the test concentrations of 0.10, 1.0 and 10% v/v saturated solution. However, growth was observed to be reduced at 100% v/v saturated solution.
Based on this information test concentrations of 10, 18, 32, 56 and 100% v/v saturated solution were selected for the definitive test.
Chemical analysis of the test preparations at 0 and 72 hours showed values of near nominal indicating that the test item was stable under test conditions.

Verification of Test Concentrations
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 11 to 110 mg/L. There was no significant change in the measured concentrations at 72 hours. As all results were within 20% of the nominal values the study results are based on the test concentrations 10, 18, 32, 56 and 100 mg/L.

Observations on Cultures
All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 10, 18 and 32 mg/L, however no intact cells were observed to be present in the test cultures at 56 and 100 mg/L.

Inhibition of Growth Rate
ErC10 (0 to 72 hour): 37 mg/L; 95% confidence limits 28 to 49 mg/L
ErC20 (0 to 72 hour): 40 mg/L; 95% confidence limits 32 to 50 mg/L
ErC50 (0 to 72 hour): 47 mg/L; 95% confidence limits 42 to 53 mg/L
Where ErCx is the test concentration that reduced growth rate by x%.
Statistical analysis of the growth rate data was carried out for the control and all test concentrations using Dunnett’s Multiple t-test incorporating Levene’s Test on Variance Homogeneity and Shapiro-Wilks test on Normal Distribution. There were no statistically significant differences between the control, 10, 18 and 32 mg/L test concentrations (P≥0.05); however, all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on growth rate was 32 mg/L. Correspondingly the LOEC based on growth rate was 56 mg/L.

Inhibition of Yield
EyC10 (0 to 72 hour): 29 mg/L; 95% confidence limits 23 to 36 mg/L
EyC20 (0 to 72 hour): 32 mg/L; 95% confidence limits 26 to 39 mg/L
EyC50 (0 to 72 hour): 40 mg/L; 95% confidence limits 34 to 48 mg/L
Where EyCx is the test concentration that reduced yield by x%.
Statistical analysis of the yield data was carried out using a Williams Multiple Sequential t-test incorporating Trend Analysis by Contrasts, Levene’s Test on Variance Homogeneity and Shapiro-Wilks test on Normal Distribution. There were no statistically significant differences between the control, 10 and 18 mg/L test concentration (P≥0.05); however, all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on yield was 18 mg/L. Correspondingly the LOEC based on yield was 32 mg/L.

Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 221 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Nominal cell density of control at 0 hours : 5.00 x 10³ cells per mL

Mean cell density of control at 72 hours : 1.11 x 10⁶ cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 11% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 to 72 hours) was 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Water quality criteria

The test item vessels showed a decrease in pH with increasing test concentration. The lowest pH recorded was 4.3 at 100 mg/L.

Temperature was maintained at 24 ±1 ºC throughout the test.

The pH value of the control cultures was observed to increase from pH 7.7 at 0 hours to pH 8.4 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 Guideline.

Observations on Test Item Solubility

At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 10, 18 and 32 mg/L test cultures were observed to be green dispersions. All 56 and 100 mg/L test cultures were observed to be clear colorless solutions.

Validity criteria fulfilled:

yes

Conclusions:

Based on the findings of the study, following results were determined as below;
Inhibition of Growth Rate
ErC10 (0 to 72 hour): 37 mg/L; 95% confidence limits 28 to 49 mg/L
ErC20 (0 to 72 hour): 40 mg/L; 95% confidence limits 32 to 50 mg/L
ErC50 (0 to 72 hour): 47 mg/L; 95% confidence limits 42 to 53 mg/L

Inhibition of Yield
EyC10 (0 to 72 hour): 29 mg/L; 95% confidence limits 23 to 36 mg/L
EyC20 (0 to 72 hour): 32 mg/L; 95% confidence limits 26 to 39 mg/L
EyC50 (0 to 72 hour): 40 mg/L; 95% confidence limits 34 to 48 mg/L

Executive summary:

A study was performed to assess the effect of the test item on the growth of the green alga Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata). The method followed that described in 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 761/2009.

Methods

Following a preliminary range-finding test, Raphidocelis subcapitata was exposed to solutions of the test item at nominal concentrations of 10, 18, 32, 56 and 100% v/v saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C. The test item solutions were prepared by stirring an excess (100 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration through a 0.2 μm Gelman Acrocap filter (first approximate 100 mL used to pre-condition the filter was discarded) to produce a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups.

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

Results

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 11 to 110 mg/L. There was no significant change in the measured concentrations at 72 hours. As all results were within 20% of the nominal values the study results are based on the test concentrations 10, 18, 32, 56 and 100 mg/L.

Exposure of Raphidocelis subcapitata to the test item gave the following results based on the measured test concentrations:

ResponseVariable	EC50 (mg/L)	95% Confidence Limits (mg/L)			No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	47	42	-	53	32	56
Yield	40	34	-	48	18	32

Description of key information

Based on the findings of the study, following results were determined as below;

Inhibition of Growth Rate

ErC50 (0 to 72 hour): 47 mg/L; 95% confidence limits 42 to 53 mg/L

Inhibition of Yield

EyC50 (0 to 72 hour): 40 mg/L; 95% confidence limits 34 to 48 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

47 mg/L

EC10 or NOEC for freshwater algae:

32 mg/L

Additional information

A study was performed to assess the effect of the test item on the growth of the green alga Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata). The method followed that described in 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 761/2009.

Methods

Following a preliminary range-finding test, Raphidocelis subcapitata was exposed to solutions of the test item at nominal concentrations of 10, 18, 32, 56 and 100% v/v saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C. The test item solutions were prepared by stirring an excess (100 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration through a 0.2 μm Gelman Acrocap filter (first approximate 100 mL used to pre-condition the filter was discarded) to produce a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups.

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

Results

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 11 to 110 mg/L. There was no significant change in the measured concentrations at 72 hours. As all results were within 20% of the nominal values the study results are based on the test concentrations 10, 18, 32, 56 and 100 mg/L.

Exposure ofRaphidocelis subcapitata to the test item gave the following results based on the measured test concentrations:

ResponseVariable	EC50(mg/L)	95% Confidence Limits (mg/L)			No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	47	42	-	53	32	56
Yield	40	34	-	48	18	32