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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
Study period:
28 Nov 2019 - 12 Mar 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was conducted in accordance with International guidelines and in accordance with GLP. All guideline validty criteria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
23 March 2006
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark and under nitrogen.
- Stability under test conditions: Assumed stable.
- Solubility and stability of the test substance in the solvent/vehicle: Treatment solutions were prepared from a water accomodated fraction (WAF) prepared at a nominal loading rate of 110 mg/L.
- Reactivity of the test substance with the test medium: Comparison of the chemical analysis values from 0 h and 72 h show that test item concentrations in the test media had declined to ca. 70 - 78 % of initial concentrations indicating that the test item was unstable in the test media.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material): Treatment slutions prepared from a WAF.

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable): N/A
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 1.0, 3.2, 10, 32 and 100 mg/L
- Sampling method: Collection
- Sample storage conditions before analysis: Frozen
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Treatment solutions prepared from water accomodated fractions (WAF) with nominal loading rates of 110, 54, 27, 14 and 6.8 mg/L. WAFs were stirred for 48 prior to the middle, dissolved fraction being decanted and filtered. The filtrates were used as the treatment solutions.
- Eluate: Algae media prepared as per OECD 201 guidance document.
- Controls: Untreated media control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/A
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): N/A
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland
- Age of inoculum (at test initiation): 3-4 days
- Method of cultivation: Constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C.

ACCLIMATION
- Acclimation period: 3-4 days
- Culturing media and conditions (same as test or not): Yes
- Any deformed or abnormal cells observed: No
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
Not reported
Test temperature:
24 ºC
pH:
8.0 - 8.6
Dissolved oxygen:
Not reported
Salinity:
Not reported
Conductivity:
Not reported
Nominal and measured concentrations:
1.0(0.0022), 3.2(0.00164), 10(0.00342), 32(0.00552), 100(0.00804) mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250ml glass conical flask
- Type (delete if not applicable): closed - plugged with polyurethane form bungs
- Material, size, headspace, fill volume: 100ml test solusoin in 250ml conical flask
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate): N/A
- Initial cells density: 5.00 x 10E3 cells/mL
- Control end cells density: 1.55 x 10E6 cels/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): N/A
- No. of vessels per vehicle control (replicates): N/A

GROWTH MEDIUM
- Standard medium used: Yes
- Detailed composition if non-standard medium was used: N/A

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Recerse osmosis
- Total organic carbon: Not reported
- Particulate matter: Not reported
- Metals: Not reported
- Pesticides: Not reported
- Chlorine: Not reported
- Alkalinity: Not reported
- Ca/mg ratio: Not reported
- Conductivity: Not reported
- Culture medium different from test medium: No
- Intervals of water quality measurement: Not reported

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: Not reported
- Photoperiod: Not reported
- Light intensity and quality: Approximatery 7000lux
- Salinity (for marine algae): N/A

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Electronic particle counter
- Chlorophyll measurement: Not determined
- Other: N/A

TEST CONCENTRATIONS
- Spacing factor for test concentrations: N/A
- Justification for using less concentrations than requested by guideline: N/A
- Range finding study: WAF
- Test concentrations: 1.0, 10, 100 mg/L Loading Rate WAF
- Results used to determine the conditions for the definitive study: 100 mg/L Loading Rate WAF
Reference substance (positive control):
yes
Remarks:
Potassium dichromate is routinely tested. Previous assessment was conducted on 16-19 July 2019 where the 72 h ErC50 was determined as 1.3 mg/L, which was within the historical normal range (a mean ErC50 value of 1.4 mg/L (standard deviation = 0.22))
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities (for algal test): No
- Unusual cell shape: Slightly enlarged cells were observed, Enlarged and misshapen cells were observed
- Colour differences: No
- Flocculation: N/A
- Adherence to test vessels: N/A
- Aggregation of algal cells: N/A
- Other: N/A
- Any stimulation of growth found in any treatment: N/A
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: N/A
- Effect concentrations exceeding solubility of substance in test medium: N/A
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: 1.3mg/L
- Other: NOEC: 0.25mg/L

Table 1 Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

Nominal Loading Rate

(mg/L)

Cell Densities* (cells per mL)

Inhibition Values (%)

0 Hours

72 Hours

Growth Rate

Yield

Control

R1

7.60E+03

1.60E+06

-

-

R2

7.63E+03

1.50E+06

Mean

7.61E+03

1.55E+06

1.0

R1

6.66E+03

1.60E+06

[1]

[3]

R2

8.04E+03

1.60E+06

Mean

7.35E+03

1.60E+06

10

R1

8.36E+03

1.39E+06

5

12

R2

9.68E+03

1.36E+06

Mean

9.02E+03

1.38E+06

100

R1

8.62E+03

1.27E+06

4

19

R2

6.75E+03

1.26E+06

Mean

7.69E+03

1.26E+06

* 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

- = Not applicable

[ ] = Increase in growth compared to controls

Table 2 Cell Densities and pH Values in the Definitive Test

Nominal Loading Rate

(mg/L)

pH

Cell Densities* (cells per mL)

pH

0 Hour

23 Hour

48 Hour

72 Hour

72 Hour

Control

R1

8.0

3.30E+04

1.65E+05

1.42E+06

8.4

R2

3.85E+04

1.95E+05

1.59E+06

R3

3.49E+04

1.96E+05

1.58E+06

R4

3.15E+04

1.68E+05

1.51E+06

R5

4.09E+04

1.78E+05

1.39E+06

R6

4.36E+04

2.09E+05

1.74E+06

Mean

3.71E+04

1.85E+05

1.54E+06

1.0

R1

8.0

3.54E+04

1.76E+05

1.53E+06

8.5

R2

3.13E+04

1.52E+05

1.42E+06

R3

3.18E+04

1.46E+05

1.38E+06

Mean

3.28E+04

1.58E+05

1.44E+06

3.2

R1

7.9

3.62E+04

1.62E+05

1.45E+06

8.5

R2

3.36E+04

1.43E+05

1.29E+06

R3

3.14E+04

1.66E+05

1.61E+06

Mean

3.38E+04

1.57E+05

1.45E+06

10

R1

7.9

2.87E+04

1.45E+05

1.40E+06

8.5

R2

2.97E+04

1.44E+05

1.37E+06

R3

3.04E+04

1.35E+05

1.52E+06

Mean

2.96E+04

1.41E+05

1.43E+06

32

R1

7.8

3.15E+04

1.51E+05

1.41E+06

8.5

R2

2.83E+04

1.20E+05

1.42E+06

R3

2.66E+04

1.34E+05

1.39E+06

Mean

2.88E+04

1.35E+05

1.41E+06

100

R1

7.8

2.95E+04

1.21E+05

1.20E+06

8.6

R2

2.52E+04

1.42E+05

1.18E+06

R3

2.40E+04

1.12E+05

1.28E+06

Mean

2.62E+04

1.25E+05

1.22E+06

* 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 3 Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Loading Rate

(mg/L)

Growth Rate (cells/mL/hour)

Yield (cells/mL)

0 to 72 Hour

% Inhibition

0 to 72 Hour

% Inhibition*

Control

R1

0.078

-

1.42E+06

-

R2

0.080

1.58E+06

R3

0.080

1.58E+06

R4

0.079

1.51E+06

R5

0.078

1.38E+06

R6

0.081

1.73E+06

Mean

0.079

1.53E+06

SD

0.001

1.26E+05

1.0

R1

0.080

[1]

1.53E+06

 

R2

0.078

1

1.41E+06

 

R3

0.078

1

1.37E+06

 

Mean

0.079

0

1.44E+06

6

SD

0.001

 

7.87E+04

 

3.2

R1

0.079

0

1.45E+06

 

R2

0.077

3

1.29E+06

 

R3

0.080

[1]

1.61E+06

 

Mean

0.079

1

1.45E+06

6

SD

0.002

 

1.59E+05

 

10

R1

0.078

1

1.40E+06

 

R2

0.078

1

1.37E+06

 

R3

0.079

0

1.52E+06

 

Mean

0.078

1

1.43E+06

7

SD

0.001

 

7.90E+04

 

32

R1

0.078

1

1.40E+06

 

R2

0.078

1

1.41E+06

 

R3

0.078

1

1.39E+06

 

Mean

0.078

1

1.40E+06

9

SD

0.000

 

1.33E+04

 

100

R1

0.076

4

1.19E+06

 

R2

0.076

4

1.17E+06

 

R3

0.077

3

1.27E+06

 

Mean

0.076

4

1.21E+06

21

SD

0.001

 

5.31E+04

 

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

R = Replicate

SD = Standard deviation

[ ] = Increase in growth compared to controls

Validity criteria fulfilled:
yes
Conclusions:
The 72 h ErL50 to green algae, under the conditions of this test was >1.7 mg/L. Statistically significant reduced growth was observed in the 1.2 and 1.7 mg/L treatment groups. The NOELR was therefore established as 0.98 mg/L.
Executive summary:

OECD 201 (2011) - In a 72 hour toxicity study, the cultures of Pseudokirchneriella subcapitata were exposed to Tetraesters of pentaerythritol with 2- methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid at measured concentrations of 0.32, 0.63, 0.98, 1.2 and 1.7 mg/L under static conditions in accordance with the OECD 201 guidance document. Treatment solutions were prepared from water accommodated fractions (WAF) with nominal loading rates of 6.8, 14, 27, 54 and 110 mg/L, respectively. The solutions were stirred for 48 h. The solubilized fractions were decanted and filtered to prepare the treatment solutions. The initial cell biomass added to each treatment flasks was 0.75x104cells/mL at 0 h. Cell biomasses were determined, using a particle counter, at 24, 48 and 72 h.

 

The ErL50for was estimated to be >1.7 mg/L, the highest tested concentration. The mean percent growth inhibition in the treated algal culture as compared to the control ranged from 0.44 – 3.3 %.

 

Statistically significant reductions in growth rate were observed, according to Dunnett’s multiple comparison test, in the 1.2 and 1.7 mg/L treatment groups (p<0.01 and p<0.01, respectively). The 72 h NOELR for growth rate was therefore determined to be 0.98 mg/L.

 

Routine microscopical examination of the algae cells concluded that there was no compound related phytotoxic effects.

 

This toxicity study is classified as acceptable and satisfies the guideline requirements for an aquatic algae toxicity study according to the OECD 201 guideline.

Description of key information

72 h ErL50: >100 mg/L; OECD 201; Hull, K. (2020)

72 h NOELR for growth rate: 100 mg/L; OECD 201; Hull, K. (2020)

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
100 mg/L

Additional information

Raphidocelis subcapitata was exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C. Analysis of the 0-Hour test preparations showed measured concentration to range from 0.0016 to 0.0080 mg/L, 72-Hour test preparations showed measured concentrations to range from 0.00058 to 0.0047 mg/L.  Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

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