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Ecotoxicological information

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:
September 2017 to May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Identification: RM10000077
Batch: 1013Q17761
Purity: 100%
Physical state/Appearance: White powder
Expiry Date: 25 June 2018
Storage Conditions: Room temperature in the dark

Sampling and analysis

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 stored frozen prior to analysis. Duplicate samples were taken at each occasion and stored frozen for further analysis if necessary.

Test solutions

Vehicle:
no
Details on test solutions:
The test substance is poorly water soluble test items. Using the approaches endorsed by ECETOC (1996) and OECD (2000) for poorly water soluble substances, the test organisms were exposed to a saturated solution of the test item. A saturated solution was prepared by stirring an excess (10 mg/l) of test item in culture medium for a period of 48 hours prior to removing any undissolved test item present by centrifugation at 40,000 g for 30 minutes to produce a 100% v/v saturated solution of the test item.

A range-finding test was performed initially by exposing the test organisms to nominal test concentrations of 0.1, 1, 10 and 100% v/v saturated solution for 72 hours.

The test concentrations for the definitive test were prepared based on the range-finding test with the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100% v/v saturated solution. A nominal amount of test item (110 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 48 hours. After 48 hours the stirring was stopped and any undissolved test item was removed by centrifugation at 40,000 g for 30 minutes to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give stock solutions of 32, 10, 3.2 and 1.0% v/v saturated solution. An aliquot (500 mL) of each of the stock solutions was separately inoculated with 2.6 mL of algal suspension to give the required test concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution. The stock solutions and each prepared concentration was inverted several times to ensure adequate mixing and homogeneity. The concentration of zinc in the test preparations was verified by chemical analysis at 0 and 72 hours

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis 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 kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C.

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 (approximately 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 104 to 105 cells/mL. The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h

Test conditions

Hardness:
No data
Test temperature:
24 +/- 1 degC
pH:
7.8 at 0 hours to 8.2 at 72 hours
Dissolved oxygen:
no data
Salinity:
not applicable
Conductivity:
no data
Nominal and measured concentrations:
Nominal test concentrations ranged from 0, 1.0, 3.2, 10, 32, and 100 % v/v saturated solution. This was equivalent to 0, 0.029, 0.078, 0.15, 0.49, and 1.4 mg/l geometric mean measured test concentrations.

The concentration of zinc in the test samples was determined and the equivalent test item concentrations calculated based on a test item zinc content of 62%. As the test media contained a source of zinc, all measured concentrations of zinc found in the test item preparations were corrected for the corresponding control level. Analysis of the test preparations at 0 hours showed measured zinc concentrations to range from less than the limit of quantification (LOQ), determined to be 0.020 mg/L, to 0.91 mg/L; equivalent to test item concentrations of less than the LOQ, determined to be 0.033 mg/L, to 1.5 mg/L. Analysis of the test preparations at 72 hours showed measured zinc concentrations to range from 0.032 to 0.86 mg/L; equivalent to test item concentrations of 0.051 to 1.4 mg/L.
Given the slight variability in the results obtained it was considered appropriate to calculate the results based on the geometric mean measured test concentrations which were determined to be 0.029, 0.078, 0.15, 0.49 and 1.4 mg/L as test item.
Details on test conditions:
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 9.74 x 105 cells per mL. Inoculation of 500 mL of test medium with 2.6 mL of this algal suspension gave an initial nominal cell density of 5.00 x 103 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 Version 2 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:
A positive control (Envigo study number RD71YQ) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.15 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
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:
other: yield
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.078 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.:
0.078 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield

Any other information on results incl. tables

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72 hour period and based on the geometric mean measured test concentrations gave the following results:

EC50 (mg/L) for growth = 0.15 mg/l

95% Confidence Limits (mg/L) for growth = 0.13 to 0.16 mg/l

No Observed Effect Concentration (NOEC) (mg/L) for growth = 0.078 mg/l

Lowest Observed Effect Concentration (LOEC) (mg/L) for growth = 0.15 mg/l

EC50 (mg/L) for yield = 0.10 mg/l

95% Confidence Limits (mg/L) for yield = no data - it was not possible to calculate 95% confidence limits for the EC50 yield value as the data did not fit the models available.

No Observed Effect Concentration (NOEC) (mg/L) for yield = 0.078 mg/l

Lowest Observed Effect Concentration (LOEC) (mg/L) for yield = 0.15 mg/l

Positive Control

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 to 72 hour) : 1.6 mg/L; 95% confidence limits 1.4 to 1.8 mg/L

EyC50 (0 to 72 hour) : 0.77 mg/L; 95% confidence limits 0.68 to 0.87 mg/L

No Observed Effect Concentration based on growth rate: 0.25 mg/L

No Observed Effect Concentration based on yield: 0.25 mg/L

Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L

Lowest Observed Effect Concentration based on yield: 0.50 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth rate of the green alga Pseudokirchneriella subcapitata was assessed using the OECD Guideline 201 and to GLP certification. The solubility of the test substance was low and an EC50 value for growth rate of 0.15 mg/l was calculated based on the geometric mean measured test concentration.
Executive summary:

The effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata was assessed using the OECD Guideline 201 and to GLP certification. Preliminary solubility work indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation. A dissolved test item concentration of approximately 1.9 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, Pseudokirchneriella subcapitata was exposed to definitive test solutions of the substance 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 (10 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 48 hours. After the stirring period any undissolved test item was removed by centrifugation at 40,000 g for 30 minutes 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.

The concentration of zinc in the test samples was determined and the equivalent test item concentrations calculated based on a test item zinc content of 62%. Analysis of the test preparations at 72 hours showed measured zinc concentrations to range from 0.032 to 0.86 mg/l; equivalent to test item concentrations of 0.051 to 1.4 mg/l. Given the slight variability in the results obtained it was considered appropriate to calculate the results based on the geometric mean measured test concentrations which were determined to be 0.029, 0.078, 0.15, 0.49 and 1.4 mg/L as test item.

The EC50 for growth rate in Pseudokirchneriella subcapitata was 0.15 mg/l based on the geometric mean measured test concentration.

The EC50 for yield in Pseudokirchneriella subcapitata was 0.10 mg/l based on the geometric mean measured test concentration.

The NOECs for both growth rate and yield in Pseudokirchneriella subcapitata were 0.078 mg/l based on the geometric mean measured test concentration.

The LOECs for both endpoints were 0.15 mg/l based on the geometric mean measured test concentration.