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Ecotoxicological information

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:
From 2017-03-27 to 2017-04-04
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:
2011
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 100, 32, 10, 3.2 and 1.0 mg test item/L, and a control
- Sampling method: Duplicate samples from the freshly prepared test media of all test concentrations and from the control were taken at the start, after 24h, 48h and at the end of the test (72h).
- Sample storage conditions before analysis: All samples were extracted directly after sampling. The extracts of the samples were stored in a fridge (4 ± 4°C) immediately after extraction, and were kept stored until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test medium of the highest test concentration of nominal 100 mg test item/L was prepared by dissolving 100.3 mg test item into 1003 mL test water by intense stirring for 10 minutes and sterilized via membrane filtration. Adequate volumes of this test medium were diluted with test water to prepare the test media of the other desired test concentrations.
- Test concentrations: 100, 32, 10, 3.2 and 1.0 mg test item/L, and a control
- Control: Test water was used without addition of the test item.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: KORSHIKOV, SAG 61.81
- Method of cultivation: The algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines.
- Source: see study report for details
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L) as CaCO3
Test temperature:
21.9 – 23.6 °C
measured daily in an Erlenmeyer flask
pH:
5.9 at test start and 6.0 to 6.2 at test end
Nominal and measured concentrations:
Nominal concentrations: 100, 32, 10, 3.2 and 1.0 mg test item/L, and a control
Measured concentrations: 57.5, 17.1, 5.68, 1.84 and 0.602 mg test item/L, and a control
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterilized Erlenmeyer flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: at least 50 mL volume containing as much test medium as possible
- Initial cells density: 5000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, As recommended in OECD TG 201

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: Continuous illumination
- Light intensity and quality: Mean light intensity: 6633 lux (range: 5900 to 7300 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometric measurement
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
36.7 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval: 30.7-43.8 mg/L
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
27.1 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
23.1 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence interval: 16.5-32.4 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
27.4 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
21 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
18.3 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
17.1 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Details on results:
- Microscopic Examination:The shape of the algal cells was not obviously affected up to this test concentration, the highest concentration tested.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: most recent test: yield 0.549 mg/L (95% CI: 0.49-0.615), growth rate 1.12 mg/L (95% CI: 1.06-1.17)
Reported statistics and error estimates:
Based on the calculated cell densities, the 72 hours ErC50 and the 72 hours EyC50, the corresponding EC20 and EC10 values and where possible their 95%-confidence limits were calculated by Probit analysis.
For the determination of the 72 hours LOEC and the 72 hours NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Williams t-test (yield) and Bonferroni-Welch t-test (growth rate).
The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

Algal densities during the test period of 72h

geometric mean test conc (mg/L) Mean algal density (10,000/mL)
24h 48h 72h
Control 1.632 5.221 34.718
0.602 2.929 8.311 42.731
1.84 3.124 6.299 39.713
5.68 2.475 6.802 36.407
17.1 3.578 5.58 33.317
57.5 2.216 0.749 0.821

Growth rates μ and percentage inhibition of μ during the test period

 

Mean measured concentrations (mg test item/L)

Growth ratesμ (1/day) and % inhibition of μ

0-24hr

0-48hr

0-72hr

μ

%

μ

%

μ

%

Control

1.144

-

1.164

-

1.410

-

0.602

1.758

-53.6

1.402

-20.5

1.482

-5.1

1.84

1.792

-56.6

1.257

-8.0

1.458

-3.4

5.68

1.595

-39.4

1.287

-10.6

1.422

-0.8

17.1

1.959

-71.2

1.190

-2.2

1.397

0.9

57.5

1.486

-29.9

0.183

84.2*

0.150

89.4*

negative values in ‘% inhibition’ indicate an increase in growth relative to that of the control
* mean value significantly different from the control (tested with Williams t-test (24 and 48h) and Bonferroni- Welch t-test (72h),α= 0.05, one-sided)

Summary of analytical results:

nominal concentration (mg/L) measured concentration (mg/L)
0h 24h 48h 72h
1 0.65 0.61 0.57 0.58
3.2 2.00 1.86 1.72 1.79
10 6.27 6.18 5.13 5.15
32 19.02 18.47 15.83 15.07
100 64.49 59.26 52.54 53.90
Validity criteria fulfilled:
yes
Conclusions:
The influence of test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The 72-hour EyC50 was calculated to be 27.4 mg test item/L, and the 72-hour ErC50 value was calculated to be 36.7 mg test item/L. The 72-hour NOEC for both, growth rate and yield, was determined to be 17.1 mg test item/L.
Executive summary:

The influence of test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test according to OECD Guideline 201.

This study encompassed 6 treatment groups (5 dose rates of the test item and a control) with three replicates per test concentration and six replicates for the control. At test start 50 mL of the test concentrations were inoculated with approx. 5000 algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72 hours for determination of cell densities by spectrophotometrical measurement.

The quantification of the test item in the test samples was performed using gas chromatography with flame ionization detector.

At the start of the test 63% of the nominal test concentrations were found (average of all test concentrations). After 24, 48 and 72 hours test duration, 55% of the nominal value was determined (average of all test concentrations). During the test the algae were exposed to a mean of 57% of nominal. Therefore, all reported results refer to mean measured concentrations.

The 72-hour EyC50 was calculated to be 27.4 mg test item/L, and the 72-hour ErC50 value was calculated to be 36.7 mg test item/L. The 72-hour NOEC for bot, growth rate and yield, was determined to be 17.1 mg test item/L.

Description of key information

The 72 -hour ErC50 value was determined to be 36.7 mg test item/L for Pseudokirchneriella subcapitata.

The 72 -hour ErC10 value was determined to be 23.1 mg test item/L for Pseudokirchneriella subcapitata.

Key value for chemical safety assessment

EC50 for freshwater algae:
36.7 mg/L
EC10 or NOEC for freshwater algae:
23.1 mg/L

Additional information

For this endpoint only one test is available. The influence of test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test according to OECD Guideline 201.

This study encompassed 6 treatment groups (5 dose rates of the test item and a control) with three replicates per test concentration and six replicates for the control. At test start 50 mL of the test concentrations were inoculated with approx. 5000 algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72 hours for determination of cell densities by spectrophotometrical measurement.

The quantification of the test item in the test samples was performed using gas chromatography with flame ionization detector.

At the start of the test 63% of the nominal test concentrations were found (average of all test concentrations). After 24, 48 and 72 hours test duration, 55% of the nominal value was determined (average of all test concentrations). During the test the algae were exposed to a mean of 57% of nominal. Therefore, all reported results refer to mean measured concentrations.

The 72 -hour ErC50, ErC10 and NOErC value were calculated to be 36.7, 23.1 and 17.1 mg test item/L, respectively.

The 72-hour EyC50, EyC10 and NOEyC were calculated to be 27.4 and 17.1 mg test item/L, respectively.