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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:

Experimental start date 26 June 2020
Experimental completion date 18 August 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Reliability 1 is assigned because the study conducted according to OECD TG 201 in compliance with GLP, without deviations that influence the quality of the results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FRET 18-0091
Chemical Name: 1-[(4,4,8-trimethyltricyclo[6.3.1.02,5]dodecan-1yl)oxy]pentan-2-ol and 1-[(1,4,4trimethyltricyclo[6.3.1.02,5]dodecan-8-yl)oxy]pentan2-ol and isomers
Batch: RDEA571-42
Purity: 92.0%
Physical State/Appearance: Light yellow viscous liquid
Expiry Date: 31 December 2021
Storage Conditions: Room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

Range finding test
A sample of each test concentration was taken for chemical analysis at 0 hours from the bulk preparations and at 72 hours from the pooled replicates in order to determine the stability of the test item under test conditions. All samples were analyzed on the day of sampling. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if required.

Definitive test
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 48 and 72 hours

Vehicle:

no

Details on test solutions:

Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.

Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals (OECD 2019).

A nominal amount of test item (1100 mg) was dispersed, in duplicate, in 11 liters of ASTM media with the aid of propeller stirring at approximately 1500 rpm for periods of either 24 or 48 hours. After stirring samples were taken for chemical analysis after the following pre-treatments:

• Centrifugation at 10000 g for 30 minutes
• Centrifugation at 40000 g for 30 minutes
• Filtration through a 0.2 μm Gelman Acrocap filter (approximately 100 mL used to pre-condition the filter was discarded)
• Filtration through a 0.2 μm Gelman Acrocap filter (approximately 500 mL used to pre-condition the filter was discarded)

Results
Stirring Period and Treatment Concentration Found (mg/L)

24 Hours Centrifuged 10000 g 1.95
24 Hours Centrifuged 40000 g 2.45
24 Hours Filtered ~ 100 mL discarded 0.618
24 Hours Filtered ~ 500 mL discarded 0.718
48 Hours Centrifuged 10000 g 4.66
48 Hours Centrifuged 40000 g 5.22
48 Hours Filtered ~ 100 mL discarded 0.647
48 Hours Filtered ~ 500 mL discarded 0.749

Discussion
Visual inspection of the centrifuged test samples showed slightly hazy dispersion had formed indicating that centrifugation as a means of removing any undissolved test item was not appropriate in this instance. Inspection of the measured concentrations obtained from the filtered test samples indicated that there was no significant increase in the dissolved test item concentration obtained when the preparation period was extended beyond 24 hours.

Based on this information the test item was prepared using a saturated solution method of preparation at an initial loading rate of 100 mg/L, stirred for a period of 24 hours prior to the removal of any undissolved test item by filtration through a 0.2 μm Gelman Acrocap filter (first approximate 500 mL discarded) to give a nominal test concentration of approximately 0.72 mg/L.

Range finding test
A nominal amount of test item (1100 mg) was dispersed in 11 liters 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 500 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 further stock solutions of 0.10, 1.0 and 10% v/v saturated solution. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (3.2 mL) to give an initial nominal cell density of 5.00 x 10^3 cells/mL.
The stock solutions and each prepared concentration were inverted several times to ensure adequate mixing and homogeneity.
The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were used for each control and test concentration.

Definitive test
A nominal amount of test item (1100 mg) was dispersed in 11 liters 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 500 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 further stock solutions of 1.0, 3.2, 10 and 32% v/v saturated solution. An aliquot (900 mL) of each of the stock solutions was separately inoculated with 8.7 mL of algal suspension to give an initial nominal cell density of 5.00 x 10^3 cells/mL.
The stock solutions and each prepared concentration were 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 10^3 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 10^5 to 10^6 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

Temperature was maintained at 24 ± 1 °C throughout the test.

pH:

The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. The pH was maintained within 7.7 - 8.0 at 0 hours and the pH was maintained within 8.1 - 8.8 at 72 hours.

Nominal and measured concentrations:

Range finding test
The range-finding test was conducted by exposing Raphidocelis subcapitata cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution for a period of 72 hours.

Definitive test
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: A series of dilutions was made from this saturated solution to give further stock solutions of 1.0, 3.2, 10 and 32% v/v saturated solution.

Chemical analysis of the fresh test preparations at 0 hours showed measured concentrations to range from 0.0061 to 0.86 mg/L. The chemical analysis of the test preparations at 72 hours showed measured concentrations to range from 0.0073 to 0.48 mg/L indicating that the test item was not stable under test conditions.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.013 to 1.1 mg/L. The measured concentrations at 24, 48 and 72 hours showed a general decline in the measured concentrations during the test to range from less than the LOQ, which was determined to be 0.010 to 0.74 mg/L at 24 hours, from less than the LOQ to 0.78 mg/L at 48 hours and from less than the LOQ to 0.80 mg/L at 72 hours. Chemical analysis of the 100% v/v saturated solution at 48 and 72 hours showed measured concentrations of 0.78 and 0.80 mg/L, respectively and the analysis of the duplicate samples yielded measured concentrations of 0.87 and 0.86 mg/L, respectively. The chemical analysis of the 3.2% saturated solution at 72 hours showed a measured concentration of 0.016 mg/L and the analysis of the duplicate sample yielded a measured concentration of 0.019 mg/L. Given that the analysis of the duplicate samples yielded similar results the original results were used for calculation purposes.

Details on test conditions:

TEST SYSTEM
- Test vessel:
The test was conducted in 250 mL glass conical flasks each containing 100 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 control group was maintained under identical conditions but not exposed to the test material.

- Initial cells density:
Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 5.20 x 10^5 cells per mL. Inoculation of 900 mL of test medium with 8.7 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10^3 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.

- No. of vessels per concentration (replicates):
2

- No. of vessels per control (replicates):
2

- No. of vessels per vehicle control (replicates):
None

CULTURE MEDIUM
The culture medium used for the preliminary media trial, range-finding and definitive tests was the same as that used to maintain the stock culture

- Standard medium used: no
- Detailed composition if non-standard medium was used:

NaNO3 25.5 mg/L
MgCl2.6H2O 12.16 mg/L
CaCl2.2H2O 4.41 mg/L
MgS04.7H20 14.6 mg/L
K2HPO4 1.044 mg/L
NaHCO3 15.0 mg/L
H3BO3 186 mg/L
MnCl2.4H20 0.415 mg/L
ZnCl2 0.00327 mg/L
FeCl3.6H2O 0.160 mg/L
CoCl2.6H2O 0.00143 mg/L
Na2MoO4.2H20 0.00726 mg/L
CuCl2.2H2O 0.000012 mg/L
Na2EDTA.2H2O 0.30 mg/L

The culture medium was prepared using reverse osmosis purified deionised water (EIga Optima 15+) and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.

- Light intensity and quality:
intensity approximately 7000 lux

- Determination of cell concentrations:
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

Reference substance (positive control):

yes

Remarks:

A positive control used potassium dichromate as the reference item at concentrations of 0.125, 0.25, 0.50, 1.0 and 2.0 mg/L. The positive control was conducted between 20 January 2020 and 06 March 2020.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.85 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.22 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.042 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Range finding test

The results showed no effect on growth at the test concentrations of 0.10 and 1.0% v/v saturated solution. However, growth was observed to be reduced at 10 and 100% v/v saturated solution.

Definitive test

Current regulatory advice is that in cases where a decline in measured concentrations is observed, geometric mean measured concentrations should be used for calculating EC50 values. It was therefore considered justifiable to base the results on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. In cases where the measured concentration was less than the LOQ of the analytical method following current regulatory advice a value of half the LOQ (i.e. 0.0050 mg/L) was used to enable calculation of the geometric mean measured concentrations.

The geometric mean measured test concentrations were determined to be:

Nominal Test Conc. (% v/v Saturated Solution) Geometric Mean Measured Test Conc. (mg/L)
1.0 0.0063
3.2 0.025
10 0.042
32 0.45
100 0.85

It is clear that the growth rate (r) and yield (y) of Raphidocelis subcapitata (CCAP 278/4) were affected by the presence of the test item.

Inhibition of Growth Rate

Statistical analysis of the growth rate data was carried out for the control and all test concentrations using William 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 (P≥0.05), between the control and 0.0063, 0.025 and 0.042 mg/L test groups; however, all other test concentrations were significantly different (P<0.05) and therefore the NOEC based on growth rate was 0.042 mg/L. Correspondingly the LOEC based on growth rate was 0.45 mg/L.

Inhibition of Yield
EyC10 (0 to 72 hour): 0.051 mg/L; 95% confidence limits 0.013 to 0.097 mg/L
EyC20 (0 to 72 hour): 0.085 mg/L; 95% confidence limits 0.030 to 0.14 mg/L
EyC50 (0 to 72 hour): 0.23 mg/L; 95% confidence limits 0.13 to 0.33 mg/L

Where EyCx is the test concentration that reduced yield by x%.

Statistical analysis of the yield data was carried out as in Section 6.2.3. There were no statistically significant differences (P≥0.05), between the control and 0.0063, 0.025 and 0.042 mg/L test groups; however, all other test concentrations were significantly different (P<0.05) and therefore the NOEC based on yield was 0.042 mg/L. Correspondingly the LOEC based on yield was 0.45 mg/L.

Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 242 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 10^3 cells per mL
Mean cell density of control at 72 hours : 1.21 x 10^6 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 7% 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%. 6.2.6

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 0.0063, 0.025, 0.042 and 0.45 mg/L, however some cell debris was observed to be present in the test cultures at 0.85 mg/L

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, 0.0063, 0.025 and 0.042 mg/L test cultures were observed to be green dispersions, whilst the 0.45 mg/L test cultures were observed to be pale green dispersions and the 0.85 mg/L test cultures remained clear colorless solutions.

Results with reference substance (positive control):

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Raphidocelis subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 to 72 hour) : 1.2 mg/L; 95% confidence limits 1.1 to 1.3 mg/L
EyC50 (0 to 72 hour) : 0.51 mg/L; 95% confidence limits 0.45 to 0.58 mg/L

No Observed Effect Concentration based on growth rate: 0.25 mg/L
No Observed Effect Concentration based on yield: 0.125 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration based on yield: 0.25 mg/L

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

Validity criteria fulfilled:

yes

Conclusions:

The ErC50, ErC10 and NOEC were 0.85, 0.22 and 0.042 mg/L

Executive summary:

Introduction

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. 1.2

Methods

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 0.72 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions. Following a preliminary range-finding test, Raphidocelis subcapitata was exposed to solutions of the test item at nominal concentrations of 1.0, 3.2, 10, 32 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 500 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. 1.3

Results

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.013 to 1.1 mg/L. Analysis of the test preparations at 24, 48 and 72 hours showed a general decline in the measured test concentrations during the test to range from less than the Limit of Quantification (LOQ), which was determined to be 0.010 mg/L to 0.74 mg/L at 24 hours, from less than the LOQ to 0.78 mg/L at 48 hours and from less than the LOQ to 0.80 mg/L at 72 hours.

Given this decline in measured test concentrations it was considered appropriate to calculate the results based on the geometric mean measured test concentrations only in order to give a “worst case” analysis of the data. The geometric mean measured test concentrations were determined to be 0.0063, 0.025, 0.042, 0.45 and 0.85 mg/L.

 

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

Response Variable	EC50 (mg/L)	95% Confidence Limits (mg/L)			No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	>0.85[1]	Not determined			0.042	0.45
Yield	0.23	0.13	-	0.33	0.042	0.45

 

[1]        It was not possible to calculate an E_rC₅₀ value as no  more than 48% inhibition of growth rate occurred at the maximum attainable dissolved test item concentration of 0.85 mg/L.

Description of key information

Introduction

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. 1.2

Methods

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 0.72 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions. Following a preliminary range-finding test, Raphidocelis subcapitata was exposed to solutions of the test item at nominal concentrations of 1.0, 3.2, 10, 32 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 500 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. 1.3

Results

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.013 to 1.1 mg/L. Analysis of the test preparations at 24, 48 and 72 hours showed a general decline in the measured test concentrations during the test to range from less than the Limit of Quantification (LOQ), which was determined to be 0.010 mg/L to 0.74 mg/L at 24 hours, from less than the LOQ to 0.78 mg/L at 48 hours and from less than the LOQ to 0.80 mg/L at 72 hours.

Given this decline in measured test concentrations it was considered appropriate to calculate the results based on the geometric mean measured test concentrations only in order to give a “worst case” analysis of the data. The geometric mean measured test concentrations were determined to be 0.0063, 0.025, 0.042, 0.45 and 0.85 mg/L.

 

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

Response Variable	EC50 (mg/L)	95% Confidence Limits (mg/L)			No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	>0.85[1]	Not determined			0.042	0.45
Yield	0.23	0.13	-	0.33	0.042	0.45

 

[1]        It was not possible to calculate an E_rC₅₀ value as no  more than 48% inhibition of growth rate occurred at the maximum attainable dissolved test item concentration of 0.85 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.85 mg/L

EC10 or NOEC for freshwater algae:

0.22 mg/L