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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:
05 March 2018 to 08 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
OECD Guideline for Testing of Chemicals, Section 2, No. 201: "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", adopted 28th July, 2011.
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
Commission Regulation (EC) No 761/2009 of 23 July 2009 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACh), Annex IV Part C, C.3 (published in the Official Journal of the European Union L 220 of 24 August 2009).
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)
Version / remarks:
EPA Ecological Effects Test Guidelines, OCSPP 850.5400, Algal Toxicity, Tiers I and II, EPA 712-C-96-164, April 1996.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
No further details specified in the study report.
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Because the Test Item was very poorly soluble in water (See more details in study with Citoxlab study code: 17/080-345AN and 17/080-316AN), a test solution was prepared using a saturated solution method according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23. A saturated Test Item solution at 100 mg/L Test Item nominal loading rate was prepared by dispersing/dissolving the needed amount of Test Item into the test medium (OECD media) two days before the start of the experiment. This solution was shaken for about 24 hours at approximately 30°C and then was equilibrated for about 24 hours at approximately 20°C. The non-dissolved test materials were removed by filtration through a fine (0.22 µm) filter to give the 100% saturated solution. As only Limit test was carried out, further dilution of this saturated solution was not performed.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:

Species: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum)
Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, and University of Göttingen, GERMANY. Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of Citoxlab Hungary Ltd.
Justification of species: The species of Pseudokirchneriella subcapitata used, being a fast-growing species, is convenient for culturing and testing and is a recommended species by relevant guidelines.
Breeding conditions: Stock cultures are small algal colonies that are inoculated onto agar regularly. These are transferred to fresh agar medium at least once every two months and are maintained under standardised conditions according to the test guidelines.
The pre-culture is intended to give a quantity of algae suitable for the inoculation of test cultures. The pre-culture was prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still growing exponentially, after an incubation period of four days. When the algal cultures contain deformed or abnormal cells, they were discarded.
Test type:
not specified
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
No post exposure observation period specified
Hardness:
Not specified
Test temperature:
The temperature was 22.5-22.7°C measured in the flask and 21.7 -23.2 °C measured within the climate chamber.
pH:
The range of the pH was 7.62 – 8.70 during the experiment.
Dissolved oxygen:
Not specified
Salinity:
Not specified
Conductivity:
Not specified
Nominal and measured concentrations:
Because significantly toxic response was not observed during the preliminary concentration Range-Finding Test, only one test concentration at 100% saturated solution and one control group were tested in a Limit Test.
Details on test conditions:
TEST CONDITIONS
Temperature
Culture temperature was checked at the beginning of the experiment and each day thereafter in a flask filled with water, in the climatic chamber. In addition, water temperature was continuously measured (with a min/max thermometer) within the climate chamber. The temperature was 22.5-22.7°C measured in the flask and 21.7 -23.2 °C measured within the climate chamber.

pH
The pH was checked at the beginning and at the end of the test, in the control and each concentration. The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 7.62 – 8.70 during the experiment.

Light Intensity
The algal culture flasks were continuously illuminated. The light intensity at the position occupied by algal culture flasks during the test was about 7813 lux (equivalent to ~106 μE/m2/s), which was ensured with fluorescent lamps (with a spectral range of 400-700 nm). The differences in light intensity between the test vessels did not exceed ± 15 % and therefore provided equal conditions for each test vessel.

DESCRIPTION OF THE TEST PROCEDURE
The exposure time was 72 hours. The test was started (0 hours) by inoculation of a biomass of approximately 104 algal cells per mL test medium.
The test was performed with 6 replicates per test concentration and 6 replicates in the control group. Volumes of 100 mL algal suspension per replicate in 250 mL Erlenmeyer flasks were continuously shaken by a laboratory orbital shaker to keep algae in suspension. The flasks were covered with air-permeable stoppers.

Preliminary Range Finding Tests
A concentration range-finding test was conducted to determine the approximate toxicity of the Test Item so that appropriate test concentrations can be selected for use in the definitive test. Algal cells were exposed to each concentration of the Test Item plus a control, for 72 hours. The test was performed with 2 replicates per each test concentration and 3 replicates in the control group.
At all applied test concentrations (including the control) the algal cells were normal, no morphological deviations were observed.

Test item concentrations in the Definitive Test
Because significantly toxic response was not observed during the preliminary concentration Range-Finding Test, only one test concentration at 100% saturated solution and one control group were tested in a Limit Test.

OBSERVATIONS
The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber. Microscopic observation of the algal cells in the test concentration and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: test item loading rate
Basis for effect:
other: growth rate, cell number & yield
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: test item loading rate
Basis for effect:
other: growth rate, cell number & yield
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: test item loading rate
Basis for effect:
other: growth rate, cell number & yield
Details on results:
VALIDITY
The cell density in the control cultures increased by the factor of 70.33 within three days.
The mean coefficient of variation for section-by-section specific growth rates (days 0-1; 1-2; 2-3) in the control cultures was 15.40 %.
The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 0.84 %.
All validity criteria were met; therefore, the study can be considered as valid.

MORPHOLOGICAL DEVIATIONS OF THE ALGAL CELLS
There were no morphological deviations of the algal cell during the study in any of the groups
Results with reference substance (positive control):
For the evaluation of the quality of the algae and validation of the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions. The date of the last study (Study Code: 17/367-022AL) with the reference item Potassium dichromate is (Batch Number: A0345704): 04–07 December 2017.
The 72h ErC 50: 0.88 mg/L, (95 % confidence limits: 0.81 – 0.96 mg/L)
The 72h EbC 50: 0.63 mg/L, (95 % confidence limits: 0.58 – 0.69 mg/L)
The 72h EyC 50: 0.53 mg/L, (95 % confidence limits: 0.49 – 0.58 mg/L)
Reported statistics and error estimates:
The results of the statistical evaluation (based on 2 Sample t-Test; α=0.05) show that the 0-72 h average specific growth rate, areas & yield was not statistically significantly different from the untreated control value, accordingly the No Observed Effect Concentration (NOEC) determined as 100% saturated solution (nominal).

Results of the Preliminary Range-Finding Test

Nominal concentrations

[test item loading rate]

Untreated control

0.1

1

10

100

Average of cell number at 72 hours

(x 104cell/mL)

69.3

70.0

68.5

69.0

67.5

 

Growth Rates (μ) and Percentage Inhibition of μ during the Test Period

Test group

Growth rate μ and % inhibition of μ

0-24 h

0-48 h

0-72 h

μ

%

μ

%

μ

%

Control

0.0589

0.0

0.0605

0.0

0.0591

0.0

100 mg/L test item

(nominal loading rate)

0.0589

0.0

0.0617

-2.0

0.0589

0.3

 

Area under the Growth Curves (A) and Percentage Inhibition of A during the Test Period

Test group

Areas under the Growth Curves (A) and % inhibition of A

0-24 h

0-48 h

0-72 h

A

%

A

%

A

%

Control

38.0

0.0

284.0

0.0

1324.0

0.0

100 mg/L test item

(nominal loading rate)

38.0

0.0

296.0

-4.2

1338.0

-1.1

 

Yield (Y) and Percentage Inhibition of Y during the Test Period

Test group

Yield (Y) and % Inhibition of Y (0-72 h)

Y

%

Control

69.3

0.0

100 mg/L test item

(nominal loading rate)

68.5

1.2

 

Cell Number (x 104cell/mL) determined in the Main Experiment

Test group

Number of cells

0 h

24 h

48 h

72 h

Control

 

1

5

16

70

 

1

4

20

73

 

1

4

20

73

 

1

3

18

67

 

1

5

16

68

 

1

4

20

71

Mean

1.00

4.17

18.33

70.33

SD

0.0

0.8

2.0

2.5

100 mg/L test item

(nominal loading rate)

 

1

3

18

66

 

1

4

19

71

 

1

4

20

73

 

1

5

20

70

 

1

5

19

68

 

1

4

20

69

Mean

1.00

4.17

19.33

69.50

SD

0.0

0.8

0.8

2.4

 

pH-Values in the Test Media at the Start and End of the Test

Test group

pH-values

Start

End

Control

7.62

8.11

8.39

8.17

8.23

8.15

8.10

100 mg/L test item

(nominal loading rate)

7.63

8.04

7.99

8.59

8.65

8.70

8.61

 

Temperature in the Climate Chamber and in the Test Media during the Test

Parameter

Exposure Time

Day 0

Day 1

Day 2

Day 3

Temperature (°C) measured in flask

22.5

22.5

22.5

22.7

Temperature (°C) of the climate chamber

Min

21.7

21.8

21.8

21.7

Max

23.1

23.1

23.2

23.2

 

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study, the ErC50, EbC50 and EyC50 values of the Test Item were determined directly from the raw data.
The 72h EbC50 value: > 100 mg/L test item nominal loading rate
The 72h ErC50 value: > 100 mg/L test item nominal loading rate
The 72h EyC50 value: > 100 mg/L test item nominal loading rate
The 72h NOEC: 100 mg/L test item nominal loading rate
The 72h LOEC: > 100 mg/L test item nominal loading rate
Based on the results of this study, the Test Item had no toxic effect at saturation; the EC50 results and the LOEC are higher than the solubility level of the Test Item in the test medium
Executive summary:

The effect of the Test Item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours.

 

Because no toxic effect was observed at the solubility level of the Test Item in the preliminary test, only one test concentration at 100% saturated solution and one Control group were tested in a Limit test in the definitive study.

 

Since the Test Item was very poorly soluble in water (solubility level in water was <LOQ in water results obtained under the following Citoxlab Hungary study code: 17/080-345AN) and due to that there was no toxic effect at the solubility level of the Test Item in the test medium during the Preliminary Range-Finding Test the experiment was carried out without analytical measurements in agreement with the Sponsor.

 

The test design included 6 replicates at test concentration and 6 replicates for the untreated control.

Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using 2 Sample t-Test (a= 0.05) by TOXSTAT software.

 

Under the conditions of the study, the ErC50, EbC50 and EyC50 values of the Test Item were determined directly from the raw data.

The 72h EbC50 value: > 100 mg/L test item nominal loading rate

The 72h ErC50 value: > 100 mg/L test item nominal loading rate

The 72h EyC50 value: > 100 mg/L test item nominal loading rate

The 72h NOEC: 100 mg/L test item nominal loading rate

The 72h LOEC: > 100 mg/L test item nominal loading rate

 

Based on the results of this study, the Test Item had no toxic effect at saturation; the EC50 results and the LOEC are higher than the solubility level of the Test Item in the test medium.

Description of key information

The 72h EbC50, ErC50, EyC50 value: > 100 mg/L test item nominal loading rate

The 72h NOEC: 100 mg/L test item nominal loading rate

The 72h LOEC: > 100 mg/L test item nominal loading rate

Key value for chemical safety assessment

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

Additional information

The effect of the Test Item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours.

Because no toxic effect was observed at the solubility level of the Test Item in the preliminary test, only one test concentration at 100% saturated solution and one Control group were tested in a Limit test in the definitive study.

Since the Test Item was very poorly soluble in water and due to that there was no toxic effect at the solubility level of the Test Item in the test medium during the Preliminary Range-Finding Test the experiment was carried out without analytical measurements in agreement with the Sponsor.

The test design included 6 replicates at test concentration and 6 replicates for the untreated control.

 

Under the conditions of the study, the ErC50, EbC50 and EyC50 values of the Test Item were determined directly from the raw data.

The 72h EbC50 value: > 100 mg/L test item nominal loading rate

The 72h ErC50 value: > 100 mg/L test item nominal loading rate

The 72h EyC50 value: > 100 mg/L test item nominal loading rate

The 72h NOEC: 100 mg/L test item nominal loading rate

The 72h LOEC: > 100 mg/L test item nominal loading rate

 

Based on the results of this study, the Test Item had no toxic effect at saturation; the EC50 results and the LOEC are higher than the solubility level of the Test Item in the test medium.