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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:
30 May – 12 Jul 2017
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:
adopted 23 March 2006, Annex 5 corrected: 28 July 2011
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesanstalt für Umwelt, Messungen und Naturschutz Baden-Württenberg, Germany
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control and 100 mg/L
- Sampling method: Samples were taken after 0,24, 48 and 72 h; All samples and the control were stabilized (1:1) with acetonitrile
- Sample storage conditions before analysis: All samples were stored deep frozen
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: test item was solved in test medium by shaking and test solutions were prepared by dilution of appropriate solutions with test medium.
- Controls: yes, medium only
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: SAG 61.81
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Semi-continuously in sterile cultures; Old medium is replaced by fresh solution periodically; new cultures are ordered regularly from commercial supplier.

ACCLIMATION
- Acclimation period: 4 days
- Culturing media and conditions: Same as test conditions.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
22.7 - 22.9 °C
pH:
7.55 - 7.65 (control)
7.51 - 7.62 (100 mg/L)
Nominal and measured concentrations:
control and 100 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
Test vessel:
- Type: Open (covered with aluminium caps)
- Material, size, headspace, fill volume: 100 mL flasks, 50 mL test solution, headspace 50 mL
- Initial cells density: 0.5 × 10 E+04 cells/mL
- Control end cells density: 19.84 × 10 E+04 cells/mL
- No. of vessels per concentration (replicates): 6 (100 mg/L)
- No. of vessels per control (replicates): 6
- CO2 supplied: By continuous agitation: Test vessels were placed in an incubator on a pivoted bogie which turns around and induces shaking by regular sudden stops.

GROWTH MEDIUM
- Standard medium used: yes (AAP medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Medium used for the test was AAP-Medium (according to Annex 3 of OECD 201).
- Intervals of water quality measurement: pH-values were measured at the start of exposure and after every 24 h.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: Continous illumination
- Light intensity and quality: 88.8 μEm-2s-1 with mean values of each platform in a range from 82.6 to 93.6 μEm-2s-1 (-7.0% to +5.4% of mean).

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Daily fluorescence measurements at an emission wavelength of 670 nm and evaluated with Tecan i-control (Software for Tecan Readers Tecan i-control, 1.11.1.0).
- Other: Microscopic evaluation of algae cells at test start and test end.

Reference substance (positive control):
yes
Remarks:
Potassium dichromate
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:
EC10
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:
other: yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
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: yes
- Observation of abnormalities: Microscopic evaluation of the cells at the start and end of exposure revealed no morphological abnormalities.
- Other: Analytical and biological results are summarized within the tables 1-5 in the section "Any other information on results incl. tables".
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 1.64 mg/L
Reported statistics and error estimates:
Statistical evaluation for the 72 hours period was performed for growth rate and yield using SAS® (2002–2010). A test for normality of the data was performed by calculating the Shapiro-Wilk statistic and the homogeneity of variance of the data was evaluated by calculating the Levene Test. The NOEC and LOEC were determined by using a multiple comparison method (Dunnetts-ttest, left sided). The calculation of the EC10, 20, 50 was not indicated due to a missing concentration response relation and since the inhibition was below 50% at the highest test item concentration for both, yield and growth rate and hence the database was inappropriate for probit analysis.

Analytical Results:

The measured initial concentration of the test item was 106% of nominal. In the aged sample the measured content was 94% of nominal. Therefore toxicological endpoints were evaluated using the nominal concentrations of the test item.

Table 1: Biological Results: Toxicological endpoints for the test item

 

Test item [mg/L]

 

nominal

ErC10 (Growth rate)

- 1)

ErC20

- 1)

ErC50

> 100

EyC10 (Yield)

- 1)

EyC20

- 1)

EyC50

> 100

NOEC 2)

100

LOEC 2)

-

1) Due to an inhibition below 50% and a missing concentration response relation the database was inappropriate for probit analysis which hence was not performed

2) Following Dunnetts-t-test (left-sided, p<0.05) for growth rate and for yield

- No LOEC was observed

Table 2: Biological Results: Percentage inhibition of growth rate

Conc.

% Inhibition of growth rate

[mg/L]

0 h – 24 h

0 h – 48 h

0 h – 72 h

Control

0.0

0.0

0.0

100

8.1

19.0

6.3

Table 3: Biological Results: Main test: Individual cell numbers

Conc.

Cell numbers [10 E+04/mL]

[mg/L]

0 h

24 h

48 h

72 h

 

0.56

1.04

2.46

13.71

 

0.58

1.35

5.23

26.21

Control

0.49

1.34

6.56

15.91

 

0.57

1.46

4.00

19.06

 

0.49

1.40

3.86

19.94

 

0.52

1.26

5.03

24.20

Mean

0.54 1)

1.31

4.52

19.84

 

0.54

1.61

4.91

23.78

 

0.54

0.99

2.38

12.02

100

0.54

1.38

1.74

11.32

 

0.54

1.18

2.67

17.40

 

0.54

1.10

3.67

16.48

 

0.54

1.18

3.23

15.81

Mean

0.54

1.24

3.10

16.14

1) The mean cell density of all control replicates is used as initial cell density for all treatment groups

Table 4: Validity 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.

36.74

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%

34%

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%.

6.8%

yes

Table 5: Analytical Results: Determined concentration of Test item

Test item nominal [mg/L]

Test item nominal [mg/L]

Sampling

Test item found [mg/L]

% of nominal

Control

0

0 h fresh

< LOD

-

 

 

72 h aged

< LOD

-

100

100

0 h fresh

106

106

 

 

72 h aged

93.5

94

- = not calculated; LOD = 0.300 mg/L test item; LOQ = 1.00 mg/L test item

Validity criteria fulfilled:
yes
Conclusions:
No effects were observed up to the test item concentration of 100 mg/L therefore the EC50 was determined to be at >100 mg/L and the NOEC at 100 mg/L.

Description of key information

EC50 (72h) >100 mg/L (P.subcapitata, OECD 201, nominal)

EC10 (72h) >100 mg/L (P.subcapitata, OECD 201, nominal)

Key value for chemical safety assessment

Additional information

One experimental study is available investigating the toxicity of the substance to aquatic freshwater algae. The study was conducted according to OECD 201 and GLP using the freshwater algae Pseudokirchneriella subcapitata (Eurofins, 2017c). The test was carried out with a test item concentration of 100 mg/L. The measured concentration of the test item at test initiation was 106% and 94% of the nominal at test end. Thus, it is justified that the effect concentrations are based on the nominal concentrations. Since no effects were observed the EC10 and EC50 for growth rate and yield were determined to be >100 mg/L.