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Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-07-23 to 2013-07-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

23 July 2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

23rd March 2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Version / remarks:

April 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

For determination of the test item concentration, samples were taken from each testing concentration and from the control at the start and at the end of the experiment.

Vehicle:

no

Details on test solutions:

Algal Mineral Salts Test Medium (dilution water):
Reconstituted algal growth medium (OECD medium, according to OECD 201 guideline) was used as dilution water in the experiment. Separate stock solutions were first prepared in deionised water (prepared in the laboratory by MILLIPORE ELIX 3 water purification system). The growth medium was prepared by adding an appropriate volume of these different stock solutions to deionised water in order to achieve the final concentrations.

Test solution:
A work solution of 100 mg/L (nominal) was first prepared by dissolving an amount of 0.03 g test item in 300 mL dilution water (OECD medium) using an overnight shaking (to obtain clear solution). For more precise preparation of the test concentrations, the work solution was further diluted to obtain a stock solution of 10 mg/L nominal concentration. The test solutions of the chosen test concentrations were prepared by appropriate dilution of this stock solution. The test solutions were prepared immediately before introduction of algae (start of the experiment).

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

- Species: Pseudokirchneriella subcapitata (formerly: Selenastrum capricornutum) (Printz-Starr).
- Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Origin: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Nikolausberger Weg 18, D-37073 Göttingen, Germany
- Breeding Conditions: The stock cultures are small algal cultures that are planted on agar regularly. These are transferred to fresh medium at least once every two months under standardised conditions according to the test guidelines.
The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture was prepared with Algal Mineral Salts Culture Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of about three days. (The pre-culture was incubated for four days at this test.)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21.7 – 22.5 °C (measured in the flask); 21.5 - 22.8 °C (measured within the climate chamber)

pH:

The pH of the control cultures varied by more than 1.5 units during the experiment. However, it was considered to be within the acceptable biological variability range and does not affect the results of the test. The range of the pH was 6.68 – 10.05 during the study.

Nominal and measured concentrations:

Nominal concentrations of test item were tested: 0.12, 0.29, 0.66, 1.51, 3.48 and 8.00 mg/L.
Measured concentrations (% of nominal) at the start of the test: 103, 93, 92
Measured concentrations (% of nominal) at the end of the test: 114, 97, 104
The analytical measurements represented that the test item concentrations remained within ± 20 % of the nominal during the test period of 72 hours, therefore the biological results are based on the nominal concentrations.

Details on test conditions:

TEST SYSTEM
- Pre-Experiments
In order to select appropriate test concentrations for use in the definitive test, a preliminary range-finding test was conducted to determine the approximate toxicity of the test item. A stock solution of 100 mg/L (nominal concentration) was first prepared by dissolving the test item in OECD medium. The test solutions were prepared by appropriate dilution of this stock solution. Algal cells were exposed to each concentration of the test item plus a control, for 72 hours. The test was performed with two replicates per each test concentration and control group.
- Test vessel: Erlenmeyer flasks of ~250 mL volume with 100 mL test medium.
- Introduction of Algae: The test was started (0 hours) by inoculation of 0.1 mL algal biomass (1E7 algal cells per mL) to 100 mL test item solution. The initial cell density was about 1E4 cells/mL in each test flask. These cells were taken from an exponentially growing pre-culture, set up three days prior to the test start using the same conditions as in the test.
- Replicates: The test was performed with three replicates at each test concentration and six replicates were included in the untreated control.

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Light Intensity: The light intensity was checked and recorded at the start of the test. The algal culture flasks were continuously illuminated. The average light intensity measured at the position occupied by algal culture flasks during the test was 7969 lux, 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:
- Determination of cell number: The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscope with counting chamber.
- Morphological changes of algal cells: The morphological changes of algal cells compared to the control were examined at 24, 48 and 72 hours after starting the test using a microscope.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.3
- Range finding study: test concentrations: 0; 0.1; 1; 10; 100 mg/L
- Results of the pre-test (% inhibition of growth rates): -; -0.1; 18.8; 100; 100

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7)

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.49 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % conf. limits 0.40 – 0.60 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.26 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % conf. limits 0.22 – 0.32mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % conf. limits 1.37 – 1.77 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.67 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % conf. limits 0.59 – 0.75 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.29 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.29 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.66 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.66 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Analytical Results
Test Concentrations:
Nominal concentrations of 0.12, 0.29, 0.66, 1.51, 3.48 and 8.00 mg/L were investigated in the main study. Concentrations of the test item were analytically determined at the start and at the end of the experiment. Samples were taken from each testing concentration and the control at the start and at the end of the test and analysed by a reverse phase HPLC method with UV detection on a LiChrospher ® 60 RP-select B column.
The three lowest test concentrations (0.12, 0.29 and 0.66 mg/L) were below the analytical measurable range. The mean measured concentrations of the test item in the three highest test concentrations (1.51, 3.48 and 8.00 mg/L) varied between 92 and 114 % of the nominal during the experiment. Based on these analytical data the obtained biological results can be related to the nominal test concentrations.

- Biological Results: All biological results are related to the nominal test item concentrations.
Growth Inhibition:
For the determination of the LOEC and NOEC, the calculated mean growth rates and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test using TOXSTAT software.
The test item had a statistically significant inhibitory effect on the growth of Pseudokirchneriella subcapitata after the exposure period of 72 hours based on the average specific growth rate and yield in the concentration range of 0.66 – 8.00 mg/L (Bonferroni t-Test, α = 0.05).
Accordingly, the 72-hour NOEC related to growth rate and yield was determined to be 0.29 mg/L.
The ErCx and EyCx values of the test item and their confidence limits were calculated using Probit analysis by TOXSTAT software.
The 72-h ErC10 was determined to be 0.49 mg/L and the 72-h ErC50 as 1.56 mg/L.
The 72-h EyC10 was determined to be 0.26 mg/L and the 72-h EyC50 as 0.67 mg/L.

Microscopic Examinations
Shape of the Algal Cells:
In the concentration range of 0.66 – 8.00 mg/L the following morphological changes were observed during the test: thin cells, smaller cells, swollen cells and pinches.

Cell Density in Control: The cell density in the control has increased from nominal N = 1E4 cells/mL at the start of the test (0 hours) to N = 84.50E4 cells/mL (mean value) after 72 hours in the in the Untreated Control.
Thus, the algal growth in the control was high enough to pass the validity criteria in this assay.

Results with reference substance (positive control):

The 72h ErC50: 1.40 mg/L, (95 % confidence limits: 1.27 – 1.54 mg/L)
The 72h EyC50: 0.78 mg/L, (95 % confidence limits: 0.70 – 0.87 mg/L)

Validity Criteria of the Study

The experiment is valid because:

- cell density in the control cultures increased by a factor of more than 16 (by a factor of 84.50) within 72 hours.

- The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72 h-tests) in the control cultures did not exceed 35 %.

- CV for section-by-section growth rate day 0-1: 9.88 %

- CV for section-by-section growth rate day 1-2: 12.47 %

- CV for section-by-section growth rate day 2-3: 9.98 %

The mean coefficient of variation for section-by-section specific growth rates: 10.78 %.

- the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7 %.

- CV for average specific growth rate day 0-3: 0.99 %

Validity criteria fulfilled:

yes

Conclusions:

The 72-h EC50 value based on growth rate and yield were determined to be 1.56 mg/L and 0.67 mg/L, respectively. The 72-h EC10 value based on growth rate and yield were determined to be 0.49 mg/L and 0.26 mg/L, respectively. The 72-h NOEC related to growth rate and yield was determined to be 0.29 mg/L.

Executive summary:

This 72-h algal growth inhibition test with Pseudokirchneriella subcapitata was conducted according to EU method C.3, OECD no.201 guideline and EPA OPPTS guideline 850.5400.

The 72-h EC50 value based on growth rate and yield were determined to be 1.56 mg/L and 0.67 mg/L, respectively. The 72-h EC10 value based on growth rate and yield were determined to be 0.49 mg/L and 0.26 mg/L, respectively. The 72-h NOEC related to growth rate and yield was determined to be 0.29 mg/L. The three lowest test concentrations (0.12, 0.29 and 0.66 mg/L) were below the analytical measurable range. The mean measured concentrations of the test item in the three highest test concentrations (1.51, 3.48 and 8.00 mg/L) varied between 92 and 114 % of the nominal during the experiment. Based on these analytical data the obtained biological results can be related to the nominal test concentrations.

Description of key information

The 72-h EC50 value based on growth rate and yield were determined to be 1.56 mg/L and 0.67 mg/L, respectively. The 72-h EC10 value based on growth rate and yield were determined to be 0.49 mg/L and 0.26 mg/L, respectively. The 72-h NOEC related to growth rate and yield was determined to be 0.29 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

1.56 mg/L

EC10 or NOEC for freshwater algae:

0.49 mg/L

Additional information

This 72-h algal growth inhibition test with Pseudokirchneriella subcapitata was conducted according to EU method C.3, OECD no.201 guideline and EPA OPPTS guideline 850.5400. The 72-h EC50 value based on growth rate and yield were determined to be 1.56 mg/L and 0.67 mg/L, respectively. The 72-h EC10 value based on growth rate and yield were determined to be 0.49 mg/L and 0.26 mg/L, respectively. The 72-h NOEC related to growth rate and yield was determined to be 0.29 mg/L. The three lowest test concentrations (0.12, 0.29 and 0.66 mg/L) were below the analytical measurable range. The mean measured concentrations of the test item in the three highest test concentrations (1.51, 3.48 and 8.00 mg/L) varied between 92 and 114 % of the nominal during the experiment. Based on these analytical data the obtained biological results can be related to the nominal test concentrations.