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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
effects on growth of green algae
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 March 2021 - 06 April 2021
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2006
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 1 mg/L
- Sampling method: Samples were taken from the solvent control and the 1.0 mg/L test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. An additional sample of the inoculated 1.0 mg/L test preparation was incubated alongside the test from which samples were taken for analysis at 24 and 48 hours. A further sample of the 1.0 mg/L test preparation containing no algal cells was incubated alongside the test from which samples were taken at 72 hours for uninoculated analysis.
- Sample storage conditions before analysis: Duplicate samples were taken on each occasion and stored frozen for further analysis if required.
Vehicle:
yes
Remarks:
Dimethylformamide
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Information provided by the Sponsor indicated that the water solubility of the test item was less than 0.050 mg/L. A preliminary media preparation trial indicated that a solvent spike method of preparation at a nominal concentration of 1.0 mg/L followed by the removal of any undissolved test item by filtration through a 0.2 µm Gelman Acrocap filter (first approximate 500 mL discarded in order to pre-condition the filter) was most appropriate for this test item.
- Controls: The control and the solvent control groups were maintained under identical conditions but not exposed to the test item. The solvent control group was exposed to 100 µL/L of dimethylformamide.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Dimethylformamide
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 100 µL/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Potential undissolved material test item was removed by filtration through a 0.2 µm Gelman Acrocap filter (first approximate 500 mL discarded in order to pre-condition the filter).
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Raphidocelis subcapitata
- Strain: The test was carried out using Raphidocelis subcapitata strain CCAP 278/4.
- Source (laboratory, culture collection): 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.
- Age of inoculum (at test initiation): Approximately 3 to 4 days before the start of the test, inoculum cultures of algae was set up at 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 105 to 106 cells/mL.

ACCLIMATION
- Culturing media and conditions (same as test or not): The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
24 ± 1 °C
pH:
For the pH values during the definitive test, see Table 3 ("Cell Densities and pH Values in the Definitive Test") in "Any other information on results incl. tables".
Nominal and measured concentrations:
Nominal: 1.0 mg/L
Measured: The geometric mean measured test concentration was determined to be 0.0075 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: The test was conducted in 250 mL glass conical flasks.
- Type (delete if not applicable): Closed, test vessels were plugged with polyurethane foam bungs to reduce evaporation.
- Material, size, headspace, fill volume: Each test vessel contained 100 mL of test preparation.
- Initial cells density: An aliquot (1 liter) of the filtered preparation was inoculated with algal suspension (4.8 mL) to give an initial nominal cell density of 5.00E+03 cells/mL.
- Control end cells density: 8.08E+5 in Control; 7.56E+05 in Solvent Control
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The culture medium was prepared using reverse osmosis purified deionized water* and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.
- Culture medium: NaNO3 25.5 mg/L; MgCl2.6H2O 12.16 mg/L; CaCl2.2H2O 4.41 mg/L; MgSO4.7H2O 14.6 mg/L; K2HPO4 1.044 mg/L; NaHCO3 15.0 mg/L; H3BO3 0.186 mg/L; MnCl2.4H2O 0.415 mg/L; ZnCl2 0.00327 mg/L; FeCl3.6H2O 0.160 mg/L; CoCl2.6H2O 0.00143 mg/L; Na2MoO4.2H2O 0.00726 mg/L; CuCl2.2H2O 0.000012 mg/L; Na2EDTA.2H2O 0.30 mg/L.
- Intervals of water quality measurement: The pH of the control, solvent control and 1.0 mg/L test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The temperature within the incubator was recorded daily. The appearance of the test media was recorded daily.

OTHER TEST CONDITIONS
- Adjustment of pH: The pH in the culture medium was adjusted to 7.5 ± 0.1 with NaOH or HCl.
- Photoperiod: Continuous illumination
- Light intensity and quality: Warm white lighting 380 to 730 nm (intensity approximately 7000 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell density was determined using a haemocytometer and light microscope at 24, 48 and 72 h.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: The range-finding test was conducted by exposing Raphidocelis subcapitata cells to a series of nominal test concentrations of 0.010, 0.10 and 1.0 mg/L for a period of 72 hours.
- Results used to determine the conditions for the definitive study: The results showed no effect on growth at the test concentrations of 0.010, 0.10 and 1.0 mg/L.

CULTURING APPARATUS
- Details on culturing apparatus used: 250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control, solvent control and 1.0 mg/L treatment groups. 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:
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.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.007 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.:
> 0.007 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.:
0.007 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): There were no abnormalities detected in any of the control or test cultures.
- Unusual cell shape: no
- Other: From the data it is clear that the growth rate (r) and yield (y) of Raphidocelis subcapitata (CCAP 278/4) were not affected by the presence of the test item at a geometric mean measured test concentration of 0.0075 mg/L over the 72-Hour exposure period.
Results with reference substance (positive control):
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.
Reported statistics and error estimates:
All statistical analyses were performed using the ToxRat computer software package (ToxRat® Solutions GMBH).

Table 1: Validation Criteria

 

Criterion from the guideline

Outcome

Validity criterion fulfilled

The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.

The cell concentration of the control cultures increased by a

factor of 162 after 72 hours and the cell concentration of the solvent control cultures

increased by a factor of 151 after 72 hours.

yes

The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%.

The mean coefficients of variation for section by section specific growth rate for the control and solvent control cultures were 14% and 11%, respectively.

yes

The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.

The coefficients of variation for average specific growth rate for the control and solvent control cultures over the test period (0 to 72 hour) were 1% and 3%, respectively.

yes

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.0082 to 0.050 mg/L (Table 2). A decline in measured test concentrations was observed in the 72-Hour test preparations to between 0.0036 and 0.022 mg/L indicating that the test item was either unstable under the conditions of the test and/or was adsorbing to the algal cells present (Table 2).

The test concentration of 0.0075 mg/L was the highest attainable test concentration that could be prepared due to the limited solubility of the test item in water and auxiliary solvent and having due regard to the amount of auxiliary solvent permitted in the study under the OECD Guideline.

Table 2: Results for Range-Finding samples

 

Time Point

 

(Hours)

Nominal Concentration of Test Item in Range-Finding Samples

Cnom

(mg/L)

Determined Concentration of Test Item in Range-Finding Samples

C

(mg/L)

Percentage of Nominal Concentration

 

(%)

0

 

 

P. Rec 1

P. Rec 2

0.010

0.10

1.0

0.000498

0.000498

0.00817

0.0339

0.00565* / 0.0502**

0.000500

0.000508

82

34

<1* / 5**

100

102

72

 

 

P. Rec 1

P. Rec 2

0.010

0.10

1.0

0.000513

0.000513

0.00360

0.0105

0.0221

0.000519

0.000546

36

11

2

101

106

Note: The concentrations quoted for the procedural recovery samples are the actual fortified concentrations mg/L. The corresponding percentage of nominal concentration values relates to the Percentage of Fortified Concentration recovered.

 

*            Sample diluted below the lowest calibration standard, therefor the duplicate frozen analyzed.

**         Duplicate sample, stored frozen prior to analysis

P. Rec= Procedural recovery

Table 3: Cell Densities and pH Values in the Definitive Test

 

Geometric Mean Measured Test Concentration (mg/L)

pH

Cell Densities* (cells per mL)

pH

0 Hours

24 Hours

46 Hours

72 Hours

72 Hours

Control

R1

7.8

3.10E+04

1.46E+05

7.60E+05

9.2

R2

3.39E+04

1.65E+05

8.42E+05

R3

3.53E+04

1.76E+05

9.03E+05

R4

3.79E+04

1.65E+05

7.62E+05

R5

3.58E+04

1.41E+05

7.21E+05

R6

3.43E+04

1.60E+05

8.63E+05

Mean

3.47E+04

1.59E+05

8.08E+05

Solvent Control

R1

7.8

3.73E+04

1.60E+05

8.41E+05

8.9

R2

3.40E+04

1.26E+05

6.64E+05

R3

2.88E+04

1.27E+05

6.60E+05

R4

3.07E+04

1.59E+05

8.16E+05

R5

3.25E+04

1.52E+05

8.31E+05

R6

2.60E+04

1.29E+05

7.25E+05

Mean

3.16E+04

1.42E+05

7.56E+05

0.0075

R1

7.7

3.22E+04

1.66E+05

8.83E+05

9.2

R2

2.97E+04

1.54E+05

7.85E+05

R3

3.34E+04

1.70E+05

9.53E+05

R4

3.29E+04

1.52E+05

7.86E+05

R5

3.16E+04

1.49E+05

8.09E+05

R6

2.86E+04

1.42E+05

7.23E+05

Mean

3.14E+04

1.55E+05

8.23E+05

 

*            Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks

R           = Replicate

Table 4: Inhibition of Growth Rate and Yield in the Definitive Test

 

Geometric Mean Measured Test Concentration

(mg/L)

Growth Rate (cells/mL/hour)

Yield (cells/mL)

0 to 72 Hour

% Inhibition

0 to 72 Hour

% Inhibition*

Control

R1

0.070

-

7.55E+05

-

R2

0.071

8.37E+05

R3

0.072

8.98E+05

R4

0.070

7.57E+05

R5

0.069

7.16E+05

R6

0.072

8.58E+05

Mean

0.071

8.03E+05

SD

0.001

7.09E+04

Solvent Control

R1

0.071

-

8.36E+05

-

R2

0.068

6.59E+05

R3

0.068

6.55E+05

R4

0.071

8.11E+05

R5

0.071

8.26E+05

R6

0.069

7.20E+05

Mean

0.070

7.51E+05

SD

0.002

8.37E+04

0.0075

R1

0.072

[3]

8.78E+05

 

R2

0.070

0

7.80E+05

 

R3

0.073

[4]

9.48E+05

 

R4

0.070

0

7.81E+05

 

R5

0.071

[1]

8.04E+05

 

R6

0.069

1

7.18E+05

 

Mean

0.071

[1]

8.18E+05

[9]

SD

0.001

 

8.20E+04

 

 

*            In accordance with the OECD test guideline only the mean value for yield is calculated

R            = Replicate

SD         = Standard Deviation

[ ]          = Increase in growth compared to solvent controls

-             = Not applicable

Validity criteria fulfilled:
yes
Remarks:
See Table 1 in "Any other information on results incl. tables"
Conclusions:
The effect of the test item on the growth of Raphidocelis subcapitata has been investigated and based on the geometric mean measured test concentration gave EC50 values of greater than 0.0075 mg/L. The No Observed Effect Concentration (NOEC) was 0.0075 mg/L. This study showed that there were no toxic effects at the limit of water solubility.

Description of key information

NOErC (72 h) ≥ 0.0075 mg/L (meas. [geom. Mean], Pseudokirchneriella subcapitata, OECD 201)

ErC10 (72 h) > 0.0075 mg/L (meas. [geom. Mean], Pseudokirchneriella subcapitata, OECD 201)

ErC50 (72 h) > 0.0075 mg/L (meas. [geom. Mean], Pseudokirchneriella subcapitata, OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:
0.007 mg/L
EC10 or NOEC for freshwater algae:
0.007 mg/L

Additional information

One GLP studies assessing the toxicity of the test item to green algae is available. The study was performed under static conditions according to OECD 201 as a limit test. The study was performed with Pseudokirchneriella subcapitata which was exposed to a nominal test item concentration of 1 mg/L for 72 h. The test substance concentration was analytically verified by HPLC. Effects on the algal growth were not observed. Based on the geometric mean measured test concentration an ErC50 (72 h) of greater than 0.0075 mg/L was determined. The NOErC (72 h) was≥0.0075 mg/L (ErC10 (72h) >0.0075 mg/L).