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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:

15 January 2016 to 20 February 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The test was conducted according to an appropriate guideline under GLP conditions. It has been assigned reliability 2 due to the non-specific analysis (TOC) of the test substance concentrations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1, 10 and 100 mg/l (WAFs)
- Sampling method: Specific analyses proved not possible and, after consultation with the sponsor, it was decided to analyse the Total Organic Carbon (TOC) concentration as a surrogate measure.
40 ml singular samples for TOC analysis were taken from all test concentrations without algae and the control without algae at t=0 h, t=24 h and t=72 h. Additionally, singular reserve samples of 40 mL were taken from all test solutions without algae for possible analysis. If not already used, these samples were stored in a refrigerator for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.
- Sample storage conditions before analysis: Samples were stored in a refrigerator until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: All test solutions were prepared separately at loading rates ranging from 1.0 to 100 mg/L. After a two-day period of magnetic stirring to reach maximum solubility of the test item in test medium, the resulting mixtures were left to stabilise for one hour, after which the Water Accommodated Fractions (WAFs) were siphoned off. All final test solutions were clear and colourless.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL.
- Controls: Test medium without test item or other additives.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): All final test solutions were clear and colourless.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation): 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/mL.

ACCLIMATION
- Acclimation period: 3-days: The pre-culture was maintained under the same conditions as used in the test.
- Culturing media and conditions (same as test or not): Yes
- Any deformed or abnormal cells observed: None reported

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Test temperature:

22-23 °C

pH:

Control: t=0 8.1, t=72 7.9
100 mg/l: t=0 8.1, t=72 7.8

Nominal and measured concentrations:

Nominal: control, 1, 10 and 100 mg/l.
The measured TOC concentration was 4.43 mg/L at the start of the test in the limit concentration, corresponding to an approximate test item concentration of 8.09 mg/L. This concentration decreased to 79% of initial after 24 hours of exposure. At the end of the test period, 45% of the initial concentration remained.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass, containing 50 mL of test solution
- Type (delete if not applicable): capped
- Material, size, headspace, fill volume: glass, 100 ml, 50 ml, 50 ml.
- Aeration: No
- Initial cells density: 1 x 104 cells/mL
- Control end cells density: 320 x10⁴ cells/mL
- No. of vessels per concentration (replicates): 6 replicates of the control and the highest test concentration;
3 replicates of each intermediate test concentration;
1 replicate of each concentration without algae for turbidity correction;
4 replicates of each concentration without algae for sampling purposes.
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (M2)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water
- Culture medium different from test medium: No
- Intervals of water quality measurement: Beginning and end of test.

OTHER TEST CONDITIONS
- Sterile test conditions: not reported
- Adjustment of pH: No
- Photoperiod: continuous
- Light intensity and quality: Continuously using TLD-lamps with a light intensity within the range of 86 to 92 μE/m2/s.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : pH measured at the beginning and at the end of the test in the control and the highest test concentration. Temperature was measured continuously.
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 20 mm). Algal medium was used as blank.
Cell densities were recorded at 24-hour intervals in the control and the limit concentration. Intermediate concentrations were measured only at the end of the exposure period.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Justification for using less concentrations than requested by guideline: combined limit/range-finding test
- Range finding study: combined limit/range-finding test
- Test concentrations: 1, 10, 100 mg/l
- Results used to determine the conditions for the definitive study: n/a: combined limit/range-finding test

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF loading rate

Basis for effect:

other: growth rate and yield

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF loading rate

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): At the end of the final test, microscopic observations were performed on the control and the limit concentration to observe for any abnormal appearance of the algae.
- Unusual cell shape: None reported
- Colour differences: None reported
- Flocculation: None reported
- Adherence to test vessels: None reported
- Aggregation of algal cells: None reported
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None reported
- Effect concentrations exceeding solubility of substance in test medium: Concentrations did not exceed the solubility of the hydrolysis products.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: The EC50 for growth rate inhibition (72h-ERC50) was 1.5 mg/L with a 95% confidence interval ranging from 1.4 to 1.6 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72h-ERC50 for the algal culture tested corresponds with this range.
The EC50 for yield decrease (72h-EYC50) was 0.51 mg/L with a 95% confidence interval ranging from 0.49 to 0.53 mg/L. The historical ranges for yield decrease lie between 0.43 and 1.1 mg/L. Hence, the 72h-EYC50 for the algal culture tested corresponds with this range.
- Other: Potassium dichromate inhibited the growth rate of this fresh water algae species at nominal concentrations of 0.56 mg/L and higher.

Reported statistics and error estimates:

Determination of the NOELR and calculation of the EL50
For determination of the NOELR and the EL50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Two-sample t-test Procedure, α=0.05, one-sided, smaller).
No EL50-values could be calculated because the test item proved to be non-toxic (EL50 > maximum soluble concentration tested).
The calculations were performed with ToxRat Professional v. 3.0.0 (ToxRat Solutions® GmbH, Germany).

Validity criteria fulfilled:

yes

Conclusions:

A 72-h EL50 of >100 mg/l (nominal, WAF loading rate) and a NOELR of 100 mg/l (nominal concentration, WAF loading rate) have been determined for the effects of the test substance on growth rate of the green algae Pseudokirchneriella subcapitata. All constituents of the substance hydrolyse rapidly in water, therefore it is likely that the algae were exposed to the hydrolysis products of the test substance.

Description of key information

Toxicity to freshwater algae: 72 hour EL₅₀ >100 mg/L and NOELR 100 mg/L (nominal, WAF loading rate) (highest concentration tested), for the effects of the test substance on growth rate of Pseudokirchneriella subcapitata, in accordance with guideline OECD 201. The rapid hydrolysis rates of the constituents of the test substance means that the algae were predominantly exposed to the hydrolysis products of the test substance.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

A 72 hour EL₅₀ value of >100 mg/L and a NOELR value of 100 mg/L (nominal, WAF loading rate) (highest concentration tested) have been determined for the effects of the submission substance (CAS 504396-15-0) on growth rate of Pseudokirchneriella subcapitata. In view of the test media preparation method/exposure regime it is likely that the test organisms were exposed predominantly to the hydrolysis products of each constituent of the tested substance.

Verification of test concentrations was carried out by Total Organic Carbon (TOC) analysis. The measured TOC concentration was 4.43 mg/L at the start of the test in the limit concentration, corresponding to an approximate test item concentration of 8.09 mg/L. This concentration decreased to 79% of initial after 24 hours of exposure. At the end of the test period, 45% of the initial concentration remained. Since TOC-analysis is a non-specific method, results are reported in terms of loading rates and expressed as mg test item per litre.