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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See "attached justification".
Reason / purpose:
read-across source
Related information:
Composition 1
Test material information:
Composition 1
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
>= 105.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Read across from analogue substance.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 105.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Read across from analogue substance.
Details on results:
The results of the read-across approach from the experimental data obtained for the supporting substance are expressed as the estimated toxicity based on molecular weights. No other adaptation is necessary.
Validity criteria fulfilled:
yes
Conclusions:
Based on the available information for the read-across approach, the target substance has a 72h-EC50 > 105.5 mg/L and a NOEC ≥ 105.5 mg/L in algae.
Executive summary:

A freshwater algae growth inhibition Limit Test was performed on the analogue substance naringin, according to OECD 201 / EU Method C.3, under GLP conditions. Based on the results of a range finding study performed at concentrations of 0.1, 1, 10, 100 mg/L test item, where no toxic response was observed, Pseudokirchneriella subcapitata was exposed to 100 mg/L test item (nominal) or medium (negative control) for 72h under static conditions. Six replicates were run both for the test and control items. Analytical monitoring was performed using a validated method, measurements were taken at the start and at the end of the test (deviation < 20%).For the evaluation of the quality of the algae and validation of the experimental conditions, a positive control is performed with potassium dichromate at least twice a year. All validity criteria were met.The 72h-EC50 of the test item in freshwater algae was determined to be > 100 mg/L (nominal, basis for effect: growth rate, yield and biomass). The overall NOEC was determined to be 105.1 mg/L (nominal) and the overall LOEC was determined to be > 100 mg/L (nominal). Based on the available information for the read-across approach, the target substance has a 72h-EC50 > 105.5 mg/L and a NOEC ≥ 105.5 mg/L in algae.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From July 17th to July 20th, 2017.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 016G050
- Expiration date of the lot/batch: 21/07/2016
- Purity test date: 21/07/2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25°C, below 70 RH%), protected from light and humidity.
- Stability under test conditions: stable during test period.
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 (control) and 100 mg/L (nominal)
- Sampling method: Analytical measurements were performed from the control and at the applied test concentration level at the beginning and at the end of experiment.
- Sample storage conditions before analysis: No storage was necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution with a nominal concentration of 100 mg/L was prepared with direct addition of the test item, mixed into the test medium (OECD Medium) using ultrasonic bath approximately 25 minutes. As a Limit Test was carried out, further dilution of stock solution was not be performed.
- Controls: A concurrent untreated control was examined in parallel to the test item concentrations. A reference control with potassium dichromate is performed at least twice a year to demonstrate satisfactory test conditions.
- 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: 61.81 SAG
- Source (laboratory, culture collection): The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, and University of Göttingen, GERMANY.
- Age of inoculum (at test initiation): 3 d.
- Method of cultivation: Cultured under standardised conditions (OECD 201) in the Ecotoxicological Laboratory of CiToxLAB Hungary Ltd. Stock cultures were small algal colonies that are inoculated onto agar regularly. These were transferred to fresh agar medium at least once every two months and were maintained under standardised conditions according to the test guidelines.The pre-culture was 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, normally after an incubation period of about three days.

ACCLIMATION
- Acclimation period: no.
- Culturing media and conditions: same as test.
- Any deformed or abnormal cells observed: no.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
22.4 – 22.6 ºC
pH:
7.44 – 8.58
Nominal and measured concentrations:
- Nominal concentrations: 0 (control), 100 mg/L.
- Measured concentrations: 0 (control); and 105 mg/L at the start and 95.8 mg/L at the end of the experiment. As the analytically measured concentration deviated not more than 20 per cent from the nominal, the biological results are based on the nominal test item concentration.
Details on test conditions:
TEST SYSTEM
- Test vessel: sterile Erlenmeyer flasks
- Type: covered by air-permeable stoppers
- Material, size, headspace, fill volume: glass, size 250 mL, fill volume 100mL test solution.
- Initial cells density: 1 E04
- Control end cells density: 70 E04
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionized water
- Culture medium different from test medium: no.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous ilumination
- Light intensity and quality: The light intensity at the position occupied by algal culture flasks during the test was about 8335 lux (equivalent to ~113 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.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: 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.
- Chlorophyll measurement: no.

TEST CONCENTRATIONS
- Range finding study: a concentration range finding study at concentrations of 0 (control), 0.1, 1, 10 and 100 mg/L was conducted to determine the approximate toxicity of the test item.
- Test concentrations: Limit Test at 100 mg/L.
- Results used to determine the conditions for the definitive study: Because no toxic effect was observed during the preliminary range-finding test, only one test concentration of the test item in the test medium (100 mg/L) and one control group were tested.
Reference substance (positive control):
yes
Remarks:
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.
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
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): no.
- Any stimulation of growth found in any treatment: no.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no.
- Effect concentrations exceeding solubility of substance in test medium: no.
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: 17/058-022AL) with the reference item Potassium dichromate is (Batch Number: A0345704): 07 – 10 March 2017.
- The 72h ErC 50: 0.88 mg/L, (95 % confidence limits: 0.81 – 0.96 mg/L)
- The 72h EyC 50: 0.54 mg/L, (95 % confidence limits: 0.50 – 0.59 mg/L)
- The 72h EbC 50: 0.64 mg/L, (95 % confidence limits: 0.59 – 0.70 mg/L)
These values are within the range of laboratory ring test data (see ISO Guideline No. 8692).
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 and yield were not statistically significantly different from the untreated control value.

Table 2. 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

0.0518

0.0

0.0604

0.0

0.0590

0.0

100 mg/L (nominal)

0.0518

0.0

0.0594

1.6

0.0586

0.6

 

Table 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

30.0

0.0

266.0

0.0

1300.0

0.0

100 mg/L (nominal)

30.0

0.0

256.0

3.8

1258.0

3.2

 

Table 4. Yield (Y) and Percentage Inhibition of Y during the Test Period

Test group

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

Y

%

Control

69.0

0.0

100 mg/L (nominal)

67.2

2.7

 

Table 5. Cell Number (× 104cell/mL) determined in the Main Experiment

Test group

 

Number of cells

 

0 h

24 h

48 h

72 h

 

Control

 

1

3

18

68

 

1

4

17

67

 

1

4

19

70

 

1

3

18

68

 

1

3

18

69

 

1

4

19

78

Mean

1.00

3.50

18.17

70.00

SD

0.0

0.5

0.8

4.0

100 mg/L (nominal)

 

1

3

17

66

 

1

4

19

70

 

1

4

18

69

 

1

3

17

68

 

1

3

16

67

 

1

4

17

69

Mean

1.00

3.50

17.33

68.17

SD

0.0

0.5

1.0

1.5

 

Table 6. pH-Values in the Test Media at the Start and End of the Test

Test group

pH-values

Start

End

Control

7.44

8.58

8.20

8.45

8.53

8.22

8.44

100 mg/L (nominal)

7.44

8.25

8.32

8.13

7.98

8.01

7.96

  

Table 7. 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 (ºC) measured in flask

22.4

22.5

22.5

22.6

temperature (ºC) of the climate chamber

max

22.6

22.6

22.9

23.1

min

22.0

22.1

22.1

22.5

Validity criteria fulfilled:
yes
Remarks:
All validity criteria were met (see 'Overall remarks'), therefore the study can be considered as valid.
Conclusions:
The 72h-EC50 of the test item in green algae Pseudokirchneriella subcapitata was found to be higher than 100 mg/L and the NOEC was determined to be 100 mg/L, both based on growth rate.
Executive summary:

A freshwater algae growth inhibition Limit Test was performed on the test item, according to OECD 201 / EU Method C.3, under GLP conditions. Based on the results of a range finding study performed at concentrations of 0.1, 1, 10, 100 mg/L test item, where no toxic response was observed, Pseudokirchneriella subcapitata was exposed to 100 mg/L test item (nominal) or medium (negative control) for 72h under static conditions. Six replicates were run both for the test and control items. Analytical monitoring was performed using a validated method, measurements were taken at the start and at the end of the test (deviation < 20%). For the evaluation of the quality of the algae and validation of the experimental conditions, a positive control is performed with potassium dichromate at least twice a year. All validity criteria were met. The 72h-EC50 of the test item in freshwater algae was determined to be > 100 mg/L (nominal, basis for effect: growth rate, yield and biomass). The overall NOEC was determined to be 100 mg/L (nominal) and the overall LOEC was determined to be > 100 mg/L (nominal).

Description of key information

Key study. Study on the analogue substance naringin, according to OECD TG 201 / EU Method C.3 (GLP study). The 72h-EC50 of the test item in freshwater algae was determined to be > 100 mg/L, the overall NOEC was determined to be 100 mg/L and the overall LOEC > 100 mg/L, all based on growth rate. Based on the available information for the read-across approach, the target substance has a 72h-EC50 > 105.5 mg/L and a NOEC ≥ 105.5 mg/L in algae.

Key value for chemical safety assessment

EC50/LC50 for freshwater algae:
100 mg/L
EC10, LC10 or NOEC for freshwater algae:
100 mg/L

Additional information

A freshwater algae growth inhibition Limit Test was performed on the analogue substance naringin, according to OECD 201 / EU Method C.3, under GLP conditions. Based on the results of a range finding study, six replicate cultures of Pseudokirchneriella subcapitata was exposed to 100 mg/L test item (nominal) or medium (negative control) for 72h under static conditions. All validity criteria were met. The 72h-EC50 of the test item in freshwater algae was determined to be > 100 mg/L (nominal, basis for effect: growth rate, yield and biomass). The overall NOEC was determined to be 100 mg/L (nominal) and the overall LOEC was determined to be > 100 mg/L (nominal). Based on the available information for the read-across approach, the target substance has a 72h-EC50 > 105.5 mg/L and a NOEC ≥ 105.5 mg/L in algae.