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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: 20 July 2017 Experimental completion date: 23 August 2017

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)

Specific details on test material used for the study:

Identification: FRET 13-0460
Chemical (IUPAC) Name: Bis(cyclohexylmethyl) ether
Physical state/Appearance: Clear colorless liquid
Storage Conditions: Approximately 4 °C in darkness

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group from the bulk test preparation at 0 hours, from samples run alongside the test at 24 and 48 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0, 24 and 72 hours and stored frozen for further analysis if necessary. Sample volumes required for chemical analysis precluded the storage of duplicate samples at 48 hours.

Vehicle:

no

Details on test solutions:

Preliminary Media Preparation Trial
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 Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions.


Range-Finding Test
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 0.43 mg/L could be obtained using a saturated solution method of preparation.
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 Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further stock solutions of 10, 1.0 and 0.10% v/v saturated solution. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (3.6 mL) to give the required test concentrations of 0.10, 1.0, 10 and
100% v/v saturated solution.
Each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

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 Sortorious Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) 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 (1500 mL) of each of the stock solutions was separately inoculated with 13.6 mL of algal suspension to give the required test concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution.
Each of the prepared concentrations 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 Pseudokirchneriella subcapitata strain CCAP 278/4. 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. 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 ± 2 ºC.
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 10^3 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 ºC until the algal cell density was approximately 10^4 - 10^5 cells/mL.
A positive control (Envigo Study Number FP48BQ) used potassium dichromate as the reference item.

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 value of the control cultures was observed to increase from pH 7.7 at 0 hours to pH 10.1 at 72 hours. This increase was considered to be due to the amount of carbon dioxide required by the large number of algal cells in the log phase of growth exceeding the transfer rate of CO2 from the gaseous phase to the aqueous phase. In this situation CO2 required for photosynthesis and growth would be derived from bicarbonate in solution which results in an increase in the pH of the culture. The increase in pH after 72 hours was in excess of that recommended in the Test Guidelines (1.5 pH units after 72 hours). This was considered to have had no adverse effect on the results of the study given that the increase in cell concentration in the control cultures exceeded the validation criterion given in the Test Guidelines.

Nominal and measured concentrations:

Range-Finding Test
nominal - 0.10, 1.0, 10 and 100% v/v

Definitive Test
nominal - 1.0, 3.2, 10, 32 and 100% v/v saturated solution
geometric mean test concentration - 0.013, 0.050 and 0.19 mg/L (10, 32 and 100% v/v)

Verification of Test Concentrations
Analysis of the 10, 32 and 100% v/v saturated solution test preparations at 0 hours (see Annex 6) showed measured test concentrations to range from 0.021 to 0.31 mg/L. A decline in measured test concentration was observed over the test duration, in the range of 0.017 to 0.20 mg/L at 24 hours, 0.011 to 0.14 mg/L at 48 hour and less than the Limit of Qunatification (LOQ) of the analytical method, determined to be 0.010 mg/L, and 0.14 mg/L at 72 hours.
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.

Details on test conditions:

Culture Medium
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture. The media used in the definitive test was prepared with the addition of 250 mg/L of sodium bicarbonate to prevent inhibition of growth due to the restriction in gaseous exchange associated with testing in an enclosed system (Herman et al 1990).

Range-Finding Test
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test. The range-finding test was conducted by exposing Pseudokirchneriella 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.
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 item.
At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.

Definitive Test
In the definitive test, 250 mL glass stoppered conical flasks were used. Six flasks each were used for the control and three flasks were used for each treatment group. All flasks were completely filled and sealed with a ground glass stopper as an additional precaution at the request of the Sponsor.
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 5.52 x 10^5 cells per mL. Inoculation of 1500 mL of test medium with 13.6 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 sealed with a ground glass stopper and incubated (INFORS Multitron incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.036 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.013 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.
Based on this information test concentrations of 1.0, 3.2, 10, 32, and 100% v/v saturated solution were selected for the definitive test.
Chemical analysis of the 10 and 100% v/v saturated solution test preparations at 0 hours showed that measured concentrations of 0.025 and 0.27 mg/L were obtained respectively. Analysis at 72 hours showed that a decline in measured concentration had been observed, indicating that the test item was unstable under test conditions.

IDefinitive test

Inhibition of Growth Rate
ErC10 (0 - 72 h): 0.036 mg/L
ErC20 (0 - 72 h): 0.053 mg/L
ErC50 (0 - 72 h): 0.10 mg/L; 95% confidence limits 0.089 – 0.12 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 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). There were no statistically significant differences between the control and the 1.0, 3.2 and 10% v/v saturated solution test groups (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 10% v/v saturated solution, equivalent to a geometric mean measured test concentration of 0.013 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 32% v/v saturated solution, equivalent to a geometric mean measured test concentration of 0.050 mg/L.

Inhibition of Yield
EyC10 (0 - 72 h): 0.018 mg/L
EyC20 (0 - 72 h): 0.025 mg/L
EyC50 (0 - 72 h): 0.047 mg/L; 95% confidence limits 0.038 – 0.057 mg/L

Where:
EyCx is the test concentration that reduced yield by x%.
There were no statistically significant differences between the control and the 1.0, 3.2 and 10% v/v saturated solution test groups (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 10% v/v saturated solution, equivalent to a geometric mean measured test concentration of 0.013 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 32% v/v saturated solution, equivalent to a geometric mean measured test concentration of 0.050 mg/L.

Results with reference substance (positive control):

A positive control (Envigo Study Number FP48BQ) 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): 1.4 mg/L; 95% confidence limits 1.2 – 1.5 mg/L
EyC50 (0 – 72 h): 0.60 mg/L; 95% confidence limits 0.52 – 0.69 mg/L

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

The geometric mean measured test concentrations were determined to be:

Nominal Test Concentration (% v/v Saturated Solution)	Geometric Mean Measured Test Concentration (mg/L)	Expressed as a Percentage of the 0-Hour Measured Test Concentration
10	0.013	63
32	0.050	69
100	0.19	62

The effect of the test item on the growth ofPseudokirchneriella subcapitatahas been investigated over a 72-Hour period and based on the geometric mean measured test concentrations gave the following results:

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	0.10	0.089	-	0.12	0.013	0.050
Yield	0.047	0.038	-	0.057	0.013	0.050

 

Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 74 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 10³cells per mL

Mean cell density of control at 72 hours       :          3.70 x 10⁵cells per mL

 

The mean coefficient of variation for section by section specific growth rate for the control cultures was 24% 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 – 72 h) was 3% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Observationson 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 1.0, 3.2, 10 and 32% v/v saturated solution, however some misshapen cells were observed to be present in the test cultures at 100% v/v saturated solution.

Water QualityCriteria

The pH value of the control cultures was observed to increase from pH 7.7 at 0 hours to pH 10.1 at 72 hours. This increase was considered to be due to the amount of carbon dioxide required by the large number of algal cells in the log phase of growth exceeding the transfer rate of CO₂from the gaseous phase to the aqueous phase. In this situation CO₂required for photosynthesis and growth would be derived from bicarbonate in solution which results in an increase in the pH of the culture. The increase in pH after 72 hours was in excess of that recommended in the Test Guidelines (1.5 pH units after 72 hours). This was considered to have had no adverse effect on the results of the study given that the increase in cell concentration in the control cultures exceeded the validation criterion given in the Test Guidelines.

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, 1.0, 3.2, 10 and 32% v/v saturated solution test cultures were observed to be green dispersions, whereas the 100% v/v saturated solution test cultures were observed to be pale green dispersions. 

 

Validity criteria fulfilled:

yes

Conclusions:

The ErC50, ErC10 and NOEC were 0.10, 0.036 and 0.013 mg/l

Executive summary:

A study was performed to assess the effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD TG No 201.

Following a preliminary range-finding test,Pseudokirchneriella subcapitatawas 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 (0.2 µm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre-condition the filter) 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.

Due to the potentially volatile nature of the test item, testing was conducted in completely filled, stoppered test vessels in order to minimize possible losses due to volatilization. following the recommendations of published data (Hermanet al1990) in order to prevent inhibition of growth due to the restriction of gaseous exchange, additional sodium bicarbonate was added to the culture medium to provide a source of carbon dioxide for algal growth.

Analysis of the 10, 32 and 100% v/v saturated solution test preparations at 0 hours showed measured test concentrations to range from 0.021 to 0.31 mg/L. A decline in measured test concentration was observed over the test duration, in the range of 0.017 to 0.20 mg/L at
24 hours, 0.011 to 0.14 mg/L at 48 hours and less than the Limit of Quantification (LOQ) of the analytical method, determined to be 0.010 mg/L, and 0.14 mg/L at 72 hours and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration only in order to give a “worst case” analysis of the data.

Exposure ofPseudokirchneriella subcapitatato 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	0.10	0.089	-	0.12	0.013	0.050
Yield	0.047	0.038	-	0.057	0.013	0.050

Description of key information

A study was performed to assess the effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD TG No 201.

Following a preliminary range-finding test,Pseudokirchneriella subcapitatawas 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 (0.2 µm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre-condition the filter) 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.

Due to the potentially volatile nature of the test item, testing was conducted in completely filled, stoppered test vessels in order to minimize possible losses due to volatilization. following the recommendations of published data (Hermanet al1990) in order to prevent inhibition of growth due to the restriction of gaseous exchange, additional sodium bicarbonate was added to the culture medium to provide a source of carbon dioxide for algal growth.

Analysis of the 10, 32 and 100% v/v saturated solution test preparations at 0 hours showed measured test concentrations to range from 0.021 to 0.31 mg/L. A decline in measured test concentration was observed over the test duration, in the range of 0.017 to 0.20 mg/L at 24 hours, 0.011 to 0.14 mg/L at 48 hours and less than the Limit of Quantification (LOQ) of the analytical method, determined to be 0.010 mg/L, and 0.14 mg/L at 72 hours and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration only in order to give a “worst case” analysis of the data.

Exposure ofPseudokirchneriella subcapitatato 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	0.10	0.089	-	0.12	0.013	0.050
Yield	0.047	0.038	-	0.057	0.013	0.050

Key value for chemical safety assessment

EC50 for freshwater algae:

0.1 mg/L

EC10 or NOEC for freshwater algae:

0.013 mg/L