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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
09 October 2018 - 22 November 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
23 March 2006, Annex 5 corrected 28 July 2011
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- The test item was handled and stored in such a way that degradation/hydrolysis and inhomogeneity were prevented.
- Solubility of the test item in water: completely miscible
Analytical monitoring:
yes
Details on sampling:
- Concentrations: from all test concentrations and the control
- Sampling method: 2.0 mL samples were taken at t=0 h, t=24 h and t=72 h from the approximate centre of the test vessels.
- Sample storage conditions before analysis: in a freezer (≤-15°C)
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test substance was completely soluble in test medium at the concentrations tested. Based on the purity of the test substance and to correct for the water content, a correction factor of 3.6 was applied. Preparation of test solutions started with the highest concentration of 100 mg/L (pure test item) applying a 15-minute period of magnetic stirring to ensure complete dissolution of the test item in test medium.The pH of the solution prepared at 100 mg/L and of the blank medium were adjusted to 6.5 using a 1 M NaOH solution (Merck, Darmstadt, Germany) as pH was found to be too low and too high in the test solution and the blank, respectively. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium.
- Controls: a blank control was included consisting of test medium without test item or other additives.
- Evidence of undissolved material: no, all test solutions were clear and colorless at the end of the preparation procedure.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata)
- Strain: NIVA CHL 1
- Source: In-house laboratory 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. Three or four 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 pre-culture was maintained under the same conditions as used in the test.

ACCLIMATION: no
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
24 mg CaCO3/L
Test temperature:
22-24°C
pH:
At the start of the test: 6.7-7.1
At the end of the test: 8.0-8.3
Nominal and measured concentrations:
Nominal: 10, 18, 32, 56 and 100 mg/L
Measured: concentrations measured over all test vessels were 94-100%, 96-100% and 97-102% of nominal concentrations at t=0, t=24 and t=72, respectively. Measured concentrations at t=24 h and t=72 h were between 96 and 105% of measured concentrations at t=0 showing that the test item concentrations remained stable during the exposure period. Based on these results, effect parameters were based on nominal test item concentrations. For details on analytical results see table 1 in 'any other information on materials and methods incl. tables'
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass with aluminium caps, perforated for ventilation, containing 50 mL of test solution
- Aeration: only natural ventilation
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 232.5 x 10^4 cells/mL
- Replicates:
3 replicates of each test concentration;
6 replicates of the control;
1 extra replicate of each test group for sampling purposes after 24 hours of exposure;
1 or 2 replicates of each test concentration without algae.

GROWTH MEDIUM
- Standard medium used: yes, M2-medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water was used to formulate the medium
- Culture medium different from test medium: yes, M1-medium was used as stock culture medium
- Intervals of water quality measurement: pH was measured at the beginning and at the end of the test, temperature of the medium was measured continuously in a temperature control vessel

OTHER TEST CONDITIONS
- Adjustment of pH: pH was adjusted when the test solutions were prepared (see 'preparation of test solutions'), the pH of the media were not further adjusted.
- Light intensity and quality: Continuously using TLD-lamps with a light intensity within the range of 80 to 81 μE.m^-2.s^-1.
- Other: Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

EFFECT PARAMETERS MEASURED:
- 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 and the extra replicates, without algae, as background for the treated solutions.
- Other: Appearance of the cells was checked at the end of the final test; microscopic observations were performed on the highest test concentration and the control to observe for any abnormal appearance of the algae.

RANGE-FINDING STUDY:
- Test concentrations: three replicates were exposed to nominal concentrations of 0.10, 1.0, 10 and 100 mg/L and a control
- Results used to determine the conditions for the definitive study: yes, a growth rate inhibition of 2.5% was observed at the highest concentration (100 mg/L) while no effects were observed at lower concentrations. Although the growth rate inhibition was biologically not relevant (<10%), the range of test concentrations for the final study (10-100 mg/L) was based on these findings.
Reference substance (positive control):
yes
Remarks:
potassium dichromate (November 2018)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: based on biological relevance
Key result
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.:
18 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: based on statistical significance
Details on results:
- Any abnormal observations: no, 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.
- Statistically significant inhibition of growth rate and yield was found at nominal concentrations of 32 mg/L and higher, resulting in 6.3% growth rate and 29% yield inhibition at the highest test concentration. However, effects on growth rate were <10%, and therefore, considered biologically not relevant. The NOEC for growth rate inhibition based on biological relevance was thus 100 mg/L.

- The experimental conditions during the study (pH and temperature) were within the limits prescribed by the study plan.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Test concentrations: 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L and a control
- 72h-ErC50: 1.05 mg/L (95% confidence interval ranging from 1.04 to 1.06 mg/L).
- Other: results were within the historical range of the test facility (0.82 - 2.6 mg/L) thereby showing that the batch of algae was adequate for testing.
Reported statistics and error estimates:
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 (Williams Multiple Sequential t-Test, α=0.05, one-sided, smaller).

The ECx-values for growth rate inhibition could not be determined since the recorded effects were below 10% (i.e. ERC10 and ERC50 > maximum concentration tested).

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

Table 2 Growth rate and percentage inhibition for the total test period

Nominal conc. (mg/L) 

Mean

Std. Dev.

n

%Inhibition

Control

1.815

0.0357

6

 

10

1.788

0.0157

3

1.5

18

1.798

0.0261

3

0.9

32

1.761

0.0166

3

3.0#

56

1.723

0.0151

3

5.1#

100

1.700

0.0288

3

6.3#

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

Validity criteria fulfilled:
yes
Remarks:
see 'overall remarks'
Conclusions:
Based on the results of a short-term aquatic toxicity study, performed according to OECD 201 and GLP principles, Substituted amino acid (2) solution inhibited growth of Pseudokirchneriella subcapitata at analytically confirmed nominal concentrations of 18 mg/L and higher. Based on biological relevance, the 72 h-NOErC was determined to be 100 mg/L. The 72 h-ErC50 and 72h-ErC10 and NOEC were determined to be >100 mg/L.
Executive summary:

The objectiveofthe study was to evaluate Substituted amino acid (2) solution FC-C 13587 for its ability to generate toxic effects in Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) during an exposure period of 72 hours and, if possible, to determine the NOEC, EC10, EC20 and EC50 for both inhibition of growth rate and inhibition of yield.

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011.

The batch of Substituted amino acid (2) solution FC-C 13587 tested was a clear colourless solution with a purity of 27.8% and was completely soluble in test medium at the concentrations tested. Based on the purity/composition of the test item, a correction factor of 3.6 was applied to compensate for the water content of the test item. All further concentrations reported are based on pure test item.

A final test was performed based on the results of a preceding range-finding test. Six replicates of exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to nominal concentrations of 10, 18, 32, 56, and 100 mg/L. The initial algal cell density was 104cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

Statistically significant inhibition of growth rate and yield was found at nominal concentrations of 32 mg/L and higher, resulting in 6.3% growth rate and 29% yield inhibition at the highest test concentration. However, effects on growth rate were <10%, and therefore, considered biologically not relevant.

Samples taken from all test concentrations and the control were analysed. The measured concentrationswere in the range of 94 – 102% relative to nominal throughout the test. Based on this result, effect parameters were expressed in terms of analytically confirmed nominal concentrations.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The 72h-EC10 and EC50 for growth rate inhibition (ErC10and ErC50) were beyond the range of concentrations tested, i.e. exceeded the regulatory limit concentration of nominally 100 mg/L.

The 72h-EC10 for yield inhibition (EYC10) was 18 mg/L with confidence intervals ranging from 10 to 26 mg/L, while the 72h-EC50 for yield inhibition (EYC50) exceeded the regulatory limit concentration of nominally 100 mg/L.

The 72h-NOEC for growth rate inhibition was 18 mg/L based on statistical significance and 100 mg/L based on biological relevance.

The 72h-NOEC for yield inhibition was 18 mg/L based on both statistical significance and biological relevance.

Description of key information

The ecotoxicity data from Substituted amino acid (2) solution is read across to Sodium salts of substituted amino acid (2). This read across is considered justified as during ecotoxicity testing, diluted test concentrations are prepared in a matrix with sufficient buffer capacity. At higher concentrations e.g. in stock solutions they are neutralized when the pH is outside the biologically optimal range. The difference between both substances is therefore considered negligible in the perspective of ecotoxicity testing.

Based on the results of a short-term aquatic toxicity study, performed according to OECD 201 and GLP principles, Substituted amino acid (2) solution inhibited growth of Pseudokirchneriella subcapitata at analytically confirmed nominal concentrations of 18 mg/L and higher. Based on biological relevance, the 72 h-NOErC was determined to be 100 mg/L. The 72 h-ErC50 and 72h-ErC10 and NOEC were determined to be >100 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
100 mg/L

Additional information

The objective of the study was to evaluate Substituted amino acid (2) solution FC-C 13587 for its ability to generate toxic effects in Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) during an exposure period of 72 hours and, if possible, to determine the NOEC, EC10, EC20 and EC50 for both inhibition of growth rate and inhibition of yield.

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011.

The batch of Substituted amino acid (2) solution FC-C 13587 tested was a clear colourless solution with a purity of 27.8% and was completely soluble in test medium at the concentrations tested. Based on the purity/composition of the test item, a correction factor of 3.6 was applied to compensate for the water content of the test item. All further concentrations reported are based on pure test item.

A final test was performed based on the results of a preceding range-finding test. Six replicates of exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to nominal concentrations of 10, 18, 32, 56, and 100 mg/L. The initial algal cell density was 104 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

Statistically significant inhibition of growth rate and yield was found at nominal concentrations of 32 mg/L and higher, resulting in 6.3% growth rate and 29% yield inhibition at the highest test concentration. However, effects on growth rate were <10%, and therefore, considered biologically not relevant.

Samples taken from all test concentrations and the control were analysed. The measured concentrationswere in the range of 94 – 102% relative to nominal throughout the test. Based on this result, effect parameters were expressed in terms of analytically confirmed nominal concentrations.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The 72h-EC10 and EC50 for growth rate inhibition (ErC10and ErC50) were beyond the range of concentrations tested, i.e. exceeded the regulatory limit concentration of nominally 100 mg/L.

The 72h-EC10 for yield inhibition (EYC10) was 18 mg/L with confidence intervals ranging from 10 to 26 mg/L, while the 72h-EC50 for yield inhibition (EYC50) exceeded the regulatory limit concentration of nominally 100 mg/L.

The 72h-NOEC for growth rate inhibition was 18 mg/L based on statistical significance and 100 mg/L based on biological relevance.

The 72h-NOEC for yield inhibition was 18 mg/L based on both statistical significance and biological relevance.