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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2006
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom, 2018
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 1.0 mg/L, solvent control
- Sampling method: Analysis at 0, 24, 48 and 72 hours. 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 immediate quantitative analysis. Two additional samples of the inoculated 1.0 mg/L test group were incubated alongside the test to provide samples for analysis at 24 and 48 hours. A single additional sample of the 1.0 mg/L test group containing no algal cells was incubated alongside the test to provide a sample for uninoculated analysis at 72 hours. A set of duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.
- Sample storage conditions before analysis: The test samples were analyzed on the day of receipt. The stability of samples stored frozen was confirmed over a storage period of 13 days.
Vehicle:
yes
Remarks:
dimethylformamide
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A preliminary media preparation trial conducted for the Daphnia magna Reproduction Test (Study Number 41202677) indicated that a solvent spike method of preparation at a nominal concentration of 1.0 mg/L was most appropriate for this test item. A nominal amount of test item (100 mg) was dissolved in dimethylformamide and the volume adjusted to 10 mL to give a 100 mg/10 mL solvent stock solution. An aliquot (200 μL) of this solvent stock solution was dispersed in 2 liters of culture medium with the aid of magnetic stirring for approximately 10 minutes to give a 1.0 mg/L stock solution. Following stirring, any undissolved test item was removed by centrifugation at 40000 g for 30 minutes. An aliquot (1 liter) of the centrifuged stock solution was inoculated with algal suspension (4.7 mL) to give the required nominal test concentration of 1.0 mg/L. The stock solution and the prepared concentration were inverted several times to ensure adequate mixing and homogeneity.
- Controls: control and solvent control (100 μL/L of dimethylformamide)
- Chemical name of vehicle: dimethylformamide
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: CCAP 278/4
- Source (laboratory, culture collection): 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.
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C.

ACCLIMATION
- Acclimation period: 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.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
24 ± 1 °C
pH:
7.9 - 8.5
Nominal and measured concentrations:
nominal: 1.0 mg/L
measured: 0.31 mg/L (geom. mean); 0.30 mg/L at 0 h, 0.32 mg/L at 24 h, 0.31 mg/L at 48 h and 0.075 mg/L at 72 h
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type (delete if not applicable): plugged with polyurethane foam stoppers
- Initial cells density: 1.0E+03 cells/mL
- Control end cells density: 7.12E+05 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6
- Other: Constant agitation by orbital shaker (approximately 150 rpm)

GROWTH MEDIUM
- Standard medium used: yes, according to guideline

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: no
- 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 temperature within the incubator was recorded daily.

OTHER TEST CONDITIONS
- Photoperiod: constant illumination
- Light intensity and quality: apprroximately 7000 lux provided by warm white lighting (380 to 730 nm)
- Other: Constant shaking at approximately 150 rpm for 72 hours

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples were taken at 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded. The appearance of the test media was recorded daily.

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: Based on the result of the range-finding (no effect on growth at the test concentrations of 0.010, 0.10 and 1.0 mg/L) test a "limit test" was conducted at a nominal concentration of 1.0 mg/L to confirm that at the limit of water solubility, no effect on algal growth was observed.
Range finding study
- Test concentrations: 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.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.31 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.:
>= 0.31 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Observation of abnormalities (for algal test): There were no abnormalities detected in any of the control, solvent control or test cultures at 72 hours.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 1.5 mg/L; 95% confidence limits 1.3 to 1.7 mg/L
Reported statistics and error estimates:
A Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) was carried out on the growth rate and yield data after 72 hours for the solvent control and the nominal 1.0 mg/L test concentration to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Analysis of an additional sample at 72 hours which had been prepared with the omission of algal cells showed a measured test concentration of 0.27 mg/L was obtained indicating that the decline in measured concentration observed was likely due to adsorption of the test item to the algal cells present rather than instability under the conditions of the test.

It was considered appropriate to base the results on the geometric mean measured test concentration using the 72-Hour test sample which contained no algal cells in order to give an accurate calculation of the concentration of test item to which the algae were exposed. The geometric mean measured test concentration was determined to be 0.31 mg/L.

Table 1: 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

0.071

 

8.03E+05

 

R2

0.066

 

5.72E+05

 

R3

0.070

 

7.64E+05

 

R4

0.071

 

8.22E+05

 

R5

0.069

 

7.31E+05

 

R6

0.065

 

5.50E+05

 

Mean

0.069

 

7.07E+05

 

SD

0.003

 

1.18E+05

 

Solvent Control

R1

0.066

 

5.94E+05

 

R2

0.067

 

6.27E+05

 

R3

0.064

 

5.11E+05

 

R4

0.067

 

6.35E+05

 

R5

0.069

 

7.13E+05

 

R6

0.067

 

5.99E+05

 

Mean

0.067

 

6.13E+05

 

SD

0.002

 

6.56E+04

 

0.31

R1

0.071

[6]

8.34E+05

 

R2

0.073

[9]

9.27E+05

 

R3

0.071

[6]

8.38E+05

 

R4

0.071

[6]

7.96E+05

 

R5

0.072

[7]

8.71E+05

[37]

R6

0.070

[4]

7.59E+05

 

Mean

0.071

[6]

8.38E+05

 

SD

0.001

 

5.82E+04

 

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

SD = Standard Deviation

[ ] = Increase in growth compared to controls

-             = Not applicable

Table 2: Analytical results of test Samples

Time Point

Nominal Concentration of

Test Item in Test Sample (cnom)

Sample Preparation Factor (F)

Determined Concentration of Test Item in Test Sample (c)

Percentage of Nominal Concentration

[hours]

[mg/L]

 

[mg/L]

[%]

0

Control

0.15

<LOQ

-

 

1.0

0.15

0.302

30

P. Rec 1

0.105

0.15

0.113

108

P. Rec 2

0.105

0.15

0.111

106

P. Rec 3

1.05

0.15

1.12

106

P. Rec 4

1.05

0.15

1.10

105

24

1.0

0.15

0.321

32

P. Rec 1

0.106

0.15

0.107

101

P. Rec 2

0.106

0.15

0.104

98

P. Rec 3

1.06

0.15

1.05

99

P. Rec 4

1.06

0.15

1.06

100

48

1.0

0.15

0.313

31

P. Rec 1

0.0977

0.15

0.0990

101

P. Rec 2

0.0977

0.15

0.0975

100

P. Rec 3

0.977

0.15

0.989

101

P. Rec 4

0.977

0.15

0.981

100

72

Control

0.15

<LOQ

-

 

1.0

0.15

0.0745

7

 

1.0 (no algae)

0.15

0.270

27

P. Rec 1

0.105

0.15

0.105

100

P. Rec 2

0.105

0.15

0.105

100

P. Rec 3

1.05

0.15

1.05

100

P. Rec 4

1.05

0.15

1.04

100


 

Table 3: Validity criteria for OECD 201.

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.

142 for control

124 for solvent control

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%

10% for control

7% for solvent control

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%.

4% for control

3% for solvent control

yes

 

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.

Description of key information

 No toxic effects up to the limit of water solubility (< 0.05 mg/L in aqua dest) for Pseudokirchneriella subcapitata (OECD 201)

Key value for chemical safety assessment

Additional information

The GLP-study with Propane-1,2,3-triyl 2-ethylhexanoate (CAS 7630-38-5) was conducted under static conditions according to OECD 201 using Pseudokirchneriella subcapitata as test organism. A limit test was conducted with 1 mg/L (nominal) test concentration. A nominal amount of test item (100 mg) was dissolved in dimethylformamide and the volume adjusted to 10 mL to give a 100 mg/10 mL solvent stock solution. An aliquot (200 μL) of this solvent stock solution was dispersed in 2 L of culture medium with the aid of magnetic stirring for approximately 10 minutes to give a 1.0 mg/L stock solution. Analysis of the 1.0 mg/L test preparations at 0, 24, 48 and 72 hours showed measured test concentrations of 0.30, 0.32, 0.31 and 0.075 mg/L, respectively. Analysis of an additional sample at 72 hours which had been prepared with the omission of algal cells showed a measured test concentration of 0.27 mg/L indicating that the decline in measured concentration observed was likely due to adsorption of the test item to the algal cells present rather than instability under the conditions of the test. Given this, it was considered appropriate to base the results on the geometric mean measured test concentration using the 72-hour test sample which contained no algal cells in order to give an accurate calculation of the concentration of test item to which the algae were exposed. The geometric mean measured test concentration was determined to be 0.31 mg/L. No growth rate related inhibition was observed in the test concentration throughout the test period of 72 h. Hence, the 72 h-EC50 was > 0.31 mg/L (geom. mean) and the 72 h-NOEC ≥ 0.31 mg/L (geom. mean). Thus, no effects up to the water solubility are expected for freshwater algae.