R-AMINR

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control at 0 and 72 hours. A volume of 1.9 mL from the approximate centre of the vessels was taken and were stored frozen until analysis.

At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.

Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at 32 mg/L but without algae (abiotic control) and samples for analysis were taken at the start and at the end of the test period.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of test solutions started with the highest concentration of 100 mg/L applying 15 minutes of magnetic stirring to accelerate dissolution of the test item in test medium. The pH of the solution was adjusted from 9.5 to 8.1 in the range-finder and from 9.6 to 8.2 in the final test using 1M HCl (Merck, Darmstadt, Germany) to lay within the optimal range for the test system. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure. 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 10^4 cells/mL. The substance was completely soluble in test medium at the concentrations tested.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In house laboratory culture
- Age of inoculum (at test initiation): Around 5 months for master culture
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

ACCLIMATION
- Acclimation period: Three 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 10^4 cells/mL. The cell density was measured immediately before use.
- Culturing media and conditions (same as test or not): The pre-culture was maintained under the same conditions as used in the test.
- Any deformed or abnormal cells observed: None

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/L

Test temperature:

Temperature maintained between 22 and 23°C

pH:

The pH was within the range 6 to 9 and did not vary by more than 1.5 units

Dissolved oxygen:

Not specified

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Main test
Nominal: control, 10, 18, 32, 56 and 100 mg/L.
Measured (0 hours): 32.9, 56.9 and 107 mg/L (at nominally 32, 56 and 100 mg/L)
Measured (72 hours): 34, 57.9 and 108 mg/L (at nominally 32, 56 and 100 mg/L)

Details on test conditions:

TEST SYSTEM
- Test vessel: All glass (100 mL)
- Type (delete if not applicable): open (aluminium caps, perforated for centilation)
- Material, size, headspace, fill volume: 100 mL vessels containing 50 mL of test solutions
- Aeration: No
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 224.9 x 10^4 cells/mL (mean)
- No. of organisms per vessel: Varies
- No. of vessels per concentration (replicates): 3 replicates
- No. of vessels per control (replicates): 6 replicates + 2 replicates of each concentration without algae.
- No. of vessels per vehicle control (replicates): NA

GROWTH MEDIUM
- Standard medium used: Yes

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: Yes
- Photoperiod: Continuous
- Light intensity and quality: Continuously using TLD-lamps with a light intensity within the range of 75 to 78 µE.m-2.s-1.
- Salinity (for marine algae): NA

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Growth rate and yield inhibition at 24, 48 and 72 hours.
- Determination of cell concentrations: 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 = 10 mm). Test medium was used as blank.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Standard as per guidelines
- Justification for using less concentrations than requested by guideline: NA
- Range finding study
- Test concentrations: control, 0.1, 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes (only minimal growth rate inhibition observed at th higest concentration of 100 mg/L).

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:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Measured concentrations:
Samples taken from nominally 32, 56, 100 mg/L and the control were analysed. The measured concentrations varied between 102 to 108% relative to the nominal values throughout the test. Based on these results, effect parameters were expressed as analytically confirmed nominal concentrations.

Results:
No significant inhibition of growth rate or yield was recorded at concentrations up to and including 56 mg/L, while growth rate and yield inhibition at 100 mg/L was statistically significant. However, growth rate inhibition was considered to be biologically not relevant at 100 mg/L, where the observed inhibition was below 10%. The NOEC based on biological relevance was thus 100 mg/L.

Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the highest test concentration when compared to the control.

Results with reference substance (positive control):

- Results with reference substance valid?
- EC50: The 72h-EC50 for growth rate inhibition (ERC50) was 1.97 mg/L with a 95% confidence interval ranging from 1.93 to 2.00 mg/L. The 72h-ERC50 was within the historical range of 0.86 and 2.30 mg/L, which is based on reference tests performed at the Test Facility during the last ten years. The 72h-ERC50 was not within the expected range of 0.92 and 1.46 mg/L as specified in ISO International Standard 8692, February 2012. Nevertheless, the test was considered valid since comparison to the most recent historical data is most indicative for sensitivity of the test system.

Reported statistics and error estimates:

For determination of the NOEC and the ECx-values 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 control revealed significant inhibition of growth rate or inhibition of yield (Williams Multiple Sequential t-test, α=0.05, one-sided, smaller).

Calculation of the EYC10 was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding concentrations of the test item.

The ERC10, EC20 and EC50-values could not be determined because the observed effects were below 10%.

ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Growth rate and percentage inhibition for the total test period:

Substance nominal concentration (mg/L)	Mean	Std. Dev.	n	% Inhibition
Control	1.804	0.0281	6
10	1.841	0.0261	3	-2.0
18	1.807	0.0162	3	-0.17
32	1.809	0.0168	3	-0.27
56	1.803	0.0432	3	0.050
100	1.718	0.0498	3	4.8#

# Effect was statistically significant however biologically not relevant (<10%).

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the test the 72 hour ErC50 was determined to be > 100 mg/L with the NOEC based on biological relevance set as 100 mg/L.

Description of key information

An EC/OECD method study is available assessing the potential toxicity of the substance to Algae. No biologically relevant effects were observed at the limit concentration in this study which provided a 72 hour ErC50 value of > 100 mg/L.  The biologically relevant NOEC was determined to be 100 mg/L which is used as the key value.

 

No data on marine water algal species are available.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

100 mg/L