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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:

March from the 14th to the 17th, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to other study

Remarks:

validated analytical method

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

adopted 23rd March, 2006

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Public Draft: April 1996

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

For determination of the test item concentration, 3 x 5 ml samples were taken from each testing concentration and from the control at the start and at the end of the experiment (on 14. and 17. March 2014). Samples were taken by pipette and were not stored but analysed immediately sampling.

Vehicle:

no

Details on test solutions:

The test solutions used in the test were prepared by mechanical dispersion without using of any solubilising agent just before the treatment. A stock solution was first prepared by dissolving an amount of 0.0679 g test item in 679 ml dilution water (OECD medium) using in order to obtain the nominal concentration of 100 mg/l.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: number 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662).
- Source: algae were supplied by the SAG: Collection of Algal Cultures, Göttingen, Germany.

ACCLIMATION
- Breeding Conditions: 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 standardized conditions according to the test guidelines.
- Pre-culturing: 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 Test Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of about three days.
- Pre-culture period: the preculture was incubated for four days at this test.
- Health check: the algal cultures used in the study did not contain deformed or abnormal cells.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.1 - 23.0 °C

pH:

7.09 - 8.24

Nominal and measured concentrations:

6.25, 12.5, 25, 50 and 100 mg/l (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks of 250 ml volume.
- Material, size, headspace, fill volume: flasks with 50 ml test medium.
- Aeration: he test flasks were covered with air-permeable stoppers.
- Shaking: treated and control algal suspensions were continuously shaken by a laboratory orbital shaker in 250 ml Erlenmeyer flasks.
- Initial cells density: the test was started (0 hours) by inoculation of 0.05 ml algal biomass (10^7 algal cells per ml) to 50 ml test item solution. The initial cell density was approximately 10^4 cells/ml in each test flask.
- No. of vessels per concentration: three replicates per test concentration.
- No. of vessels per control: dilution water (OECD medium) without addition of test item, six replicates.

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water in the experiment. Separate stock solutions were first prepared in deionised water. 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.
- Stock solution 1 (macro nutrients): NH4Cl 15.0 mg/, MgCl2 (anhydrous) 5.6 mg/l (this corresponds to the concentration of 12.0 mg/l of MgCl2 × 6 H2O (cf. OECD no. 201 guideline)), CaCl2 × 2 H2O 18.0 mg/l, MgSO4 × 7 H2O 15.0 mg/l, KH2PO4 1.6 mg/l.
- Stock solution 2 (iron): FeCl3 × 6 H2O 64.0 μg/l, Na2EDTA × 2 H2O 100.0 μg/l.
- Stock solution 3 (trace elements): H3BO3 185.0 μg/l, MnCl2 × 4 H2O 415.0 μg/l, ZnCl2 3.0 μg/l, CoCl2 × 6 H2O 1.5 μg/l, CuCl2 × 2 H2O 0.01 μg/l, Na2MoO4 × 2 H2O 7.0 μg/l.
- Stock solution 4 (bicarbonate): NaHCO3 50.0 mg/l.

OTHER TEST CONDITIONS
- Photoperiod: continuously illuminated with fluorescent lamps (with a spectral range of 400-700 nm).
- Light intensity: average measured light intensity was 8928 lux. 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 concentration was determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber.
- Other: the cell morphology was examined in parallel.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: one control and five concentrations in a geometric series differing by a constant factor of 2.0 were included in the main experiment.
- Justification for choice: the chosen concentrations are based on the results of the preliminary range finding tests.

RANGE-FINDING STUDY
- Test type: two non-GLP preliminary test.
- Test concentrations: 0.1; 1; 10; 50 and 100 mg/l having 100 ml test solution / test unit. As the test item is a strongly coloured substance, a second pre-test was performed at the test item concentrations of 10; 50 and 100 mg/l.
- Test system: in this second preliminary study, the light path was shortened by reduction of volume of test solutions to 50 ml / test unit.
- Duration: 72 hours.
- No. of vessels per concentration: both the first and the second pre-test were performed with two replicates in the treatment groups.
- Control: the first pre-test was performed with three replicates in the control. The second pre-test was performed with two replicates in the control groups.
- Results used to determine the conditions for the definitive study:
First pre-test: % Inhibition of growth rate was -2.3 at 01 mg/l, -0.2 at 1 mg/l, 5.5 at 10 mg/l, 16.5 at 50 mg/l and 35.3 at 100 mg/l.
First pre-test: % Inhibition of yield was -6.5 at 01 mg/l, 1.7 at 1 mg/l, 21.0 at 10 mg/l, 47.9 at 50 mg/l and 75.4 at 100 mg/l.
Second pre-test: % Inhibition of growth rate was 8.3 at 10 mg/l, 11.1 at 50 mg/l and 13.7 at 100 mg/l.
Second pre-test: % Inhibition of yield was 28.5 at 10 mg/l, 35.9 at 50 mg/l and 42.2 at 100 mg/l.

REFERENCE COMPOUND
For the evaluation of the reliability of the applied test system and the experimental conditions potassium dichromate is tested at least twice a year.
The date of the last study (Study Code: 392.440.4633) with the reference item Potassium dichromate was: 01 – 04 October 2013.

VALIDITY CRITERIA
For the test to be valid, the following performance criteria should be met:
- The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period. This corresponds to a specific growth rate of 0.92 day-1.
- 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 %.
- 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.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

130.18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 88.62 – 191.23 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

27.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 24.09 – 31.63 mg/L

Details on results:

After the exposure period of 72 hours average specific growth rates and yield were significantly different from that of the control group in the concentrations of 12.5; 25; 50 and 100 mg/l.
NOEC (72h) growth rate and yield: 6.25 mg/l.
LOEC (72h) growth rate and yield: 12.5 mg/l.
The behaviour of the test item did not show any remarkable observations. In the test morphological effects were not observed at the test item concentrations.
The shape of the algal cells growing was obviously not affected by the test item.

CONTROL
The cell density in the control has increased from nominal N= 1 x 10^4 cells/ml at the start of the test (0 hours) to N= 71.33 x 10^4 cells/ml (mean value) after 72 hours by a factor of 71.33. Thus, the algal growth in the control was high enough to pass the validity criteria in this assay.
In the test morphological effects were not observed neither in the control.

TEST CONCENTRATIONS
The analytically measured test item concentrations were 5.71; 11.61; 22.81; 47.23 and 91.53 mg/l at the start and 5.93; 12.23; 23.84; 50.77 and 99.36 mg/l at the end of the test. The analytically measured test item concentrations ranged between 91 – 94 % of the nominal at the start and 95 – 102 % of the nominal at the end of the test. As the performed analytical determinations confirmed that the test item concentration remained within the range of ±20 % of the nominal concentration, the biological results are reported based on the nominal concentration of the test item.

Results with reference substance (positive control):

ErC50 (72h): 1.33 mg/l, (95 % confidence limits: 1.22 - 1.46 mg/l)
EyC50 (72h)0: 0.90 mg/l, (95 % confidence limits: 0.81 - 1.00 mg/l)

Validity criteria fulfilled:

yes

Remarks:

density in the control cultures increased by a factor of 71.33 within 72 hours, the mean coefficient of variation for section-by-section specific growth rates was 5.90 % and specific growth rates during the test period in the control cultures was 1.68 %

Conclusions:

ErC50 (72h): 130.18 mg/l (nominal)
EyC50 (72h): 27.60 mg/l (nominal)

Executive summary:

The effect of test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours on the recommendations of theOECD Guideline for Testing of Chemicals, No. 201. Based on the results of two non-GLP preliminary tests, the nominal test item concentrations included in the study were 6.25; 12.5; 25; 50 and 100 mg/l.The analytically measuredmtest item concentrations ranged between 91 – 94 % of the nominal at the start and 95 – 102 % of the nominal at the end of the test. As the performed analytical determinations confirmed that the test item concentration remained within the range of 20 % of the nominal concentration, the biological results are reported based on the nominal concentrations of the test item.

With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rate and yield were significantly lower from that of the control group in the concentration range of 12.5 – 100 mg/l (Bonferroni t-Test; a=0.05).

Based on the statistical and biological evaluation of the test, the overall NOEC was determined to be 6.25 mg/l. The overall LOEC of the test item is 12.5 mg/l.

Conclusion

ErC50 (72h): 130.18 mg/l (nominal)

EyC50 (72h): 27.60 mg/l (nominal)

Description of key information

Not harmful/toxic to aquatic algae and cyanobacteria.

Key value for chemical safety assessment

EC50 for freshwater algae:

130 mg/L

EC10 or NOEC for freshwater algae:

6.25 mg/L

Additional information

The effect of test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), according to the OECD Guideline for Testing of Chemicals, No. 201. The analytically measured test item concentrations ranged between 91 – 94 % of the nominal at the start and 95 – 102 % of the nominal at the end of the test. With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rate and yield were significantly lower from that of the control group in the concentration range of 12.5 – 100 mg/l (Bonferroni t-Test; a=0.05). Based on the statistical and biological evaluation of the test, the overall NOEC was determined to be 6.25 mg/l. The overall LOEC of the test item is 12.5 mg/l.