Reaction mass of calcium 2,6-bis(3-carboxylatopropanamido)hexanoate and isomers of calcium amino-(3-carboxylatopropanamido)hexanoate

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:

23 March 2017 - 20 June 2017

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)

Version / remarks:

23 March, 2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

1 March 2016

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:

Three parallel samples were taken from each test concentration and from the control on both days of the analytical occasion.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The test solutions used in the test were prepared by mechanical dispersion without the aid of any solubilising agents. The test solutions were freshly prepared at the beginning of the experiment. A stock solution of 100 mg/L (nominal concentration) was prepared by dissolving 0.1030 g of the test item in 1030 mL OECD medium. The further test solutions were prepared by appropriate dilution of this stock solution.

- Controls:
Untreated Control: OECD Medium (without test item) was inoculated and examined in parallel in the study.
Toxic Reference Control: For the evaluation of the quality of the algae and the experimental conditions, potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source (laboratory, culture collection): supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany
- Breeding conditions: The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture was prepared with OECD 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

Remarks:

OECD Medium, according to OECD 201

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

no

Test temperature:

22.6 – 23.3 °C measured in the flask and 21.9 – 24.0 °C measured within the climate chamber

pH:

7.85 – 8.85

Nominal and measured concentrations:

nominal: 1.024, 2.56, 6.4, 16, 40, 100 mg/L (spacing factor for test concentrations: 2.5)
measured: 1.018, 2.44, 6.69, 15.3, 38.4, 96.7 mg/L (at start of experiment) and 0.948, 2.41, 6.11, 15.4, 38.9, 98.4 mg/L (at the end of experiment)
There is evidence that the concentration of the tested substance was maintained within ± 20 % of the nominal concentration throughout the test.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks of ~250 mL volume, Volumes of 100 mL algal suspension per replicate were continuously shaken by a lab
oratory orbital shaker in flasks for an exposure time of 72 hours
- Type: closed, flasks were covered with air-permeable stoppers
- Initial cells density: The test was started (0 hours) by inoculation of 0.1 mL algal biomass (1E07 algal cells per mL) to 100 mL test item solution. The initial cell density was about 1E04 cells/ mL in each test flask.
- Control end cells density: 63.67x1E04 cells/mL after 72 hours
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/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/L, MgCl2 (anhydrous) 5.6 mg/L, 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 × 2H2O 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
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: The algal culture flasks were continuously illuminated.
- Light intensity and quality: The average light intensity measured at the position occupied by algal culture flasks during the test was 7998 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 concentrations: 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.
- Other: 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: 2.5
- Range finding study
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 prepared by dissolving the test item in OECD medium. The further test solutions were prepared by appropriate dilution of this stock solution. Untreated control ran parallel in the test. Algal cells were exposed to each concentration of the test item plus control, for 72 hours. The test was performed with two replicates in treatment groups and three replicates in the control.
- Test concentrations: Based on the results of the Range-Finding Test six test concentrations in a geometric series were used in the main test. Nominal: 1.024, 2.56, 6.4, 16, 40, 100 mg/L (spacing factor for test concentrations: 2.5) and measured: 1.018, 2.44, 6.69, 15.3, 38.4, 96.7 mg/L (at start of experiment) and 0.948, 2.41, 6.11, 15.4, 38.9, 98.4 mg/L (at the end of experiment)

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7)

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

26.79 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

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:

EC50

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.:

2.56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

6.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.97 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

8.78 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

69.91 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

6.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

The test item had a statistically significant inhibitory effect on the growth of Raphidocelis subcapitata after the exposure period of 72 hours based on the average specific growth rate and yield at the concentrations of 6.4, 16, 40 and 100 mg/L (Dunnett’s Test, 2 tailed, α=0.05) when compared to the control. Accordingly, the 72-hour NOEC based on growth rate and yield was determined to be 2.56 mg/L and the LOEC as 6.4 mg/L.
- Exponential growth in the control: yes
- Observation of abnormalities:
No morphological abnormalities were observed in the control and in the lowest test item concentration (1.024 mg/L) during the experiment. Smaller cells (and also some swollen cells among cells without sign of any effect) were detected in the test groups of 2.56 and 6.4 mg/L. In the test groups of 16, 40 and 100 mg/L swollen, thin and small cells (together with cells without sign of any effect) concentrated in groups (approx. 20 cells) were observed.
- Unusual cell shape: yes
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none

Results with reference substance (positive control):

The 72h ErC50: 0.99 mg/L, (95 % confidence limits: 0.68 – 1.49 mg/L)
The 72h EyC50: 0.59 mg/L, (95 % confidence limits: 0.47 – 0.75 mg/L)

Reported statistics and error estimates:

Mean values and standard deviations of cell concentrations were calculated for each treatment at the start, 24 h and 48 h and at the end of the test (72 hours after the start of the test) using Microsoft Excel for Windows. Percentage inhibition of growth rate (μ) and yield (y) were calculated using EXCEL for Windows.
The ErCx, and EyCx values of the test item and their confidence limits were calculated using Probit analysis by SPSS+ software.
For the determination of the LOEC and NOEC, the calculated mean growth rate (μ) and yield (y) at the test concentrations were tested on significant differences to control values by Dunnett’s Test using SPSS+.

Validity criteria fulfilled:

yes

Conclusions:

In an 72-h algal growth inhibition test with Raphidocelis subcapitata, the obtained results showed that the test item had significant toxic effects on the growth of Raphidocelis subcapitata. The 72-hour NOEC based on growth rate and yield was determined to be 2.56 mg/L and the LOEC as 6.4 mg/L. The EC10 and EC50 based on growth rate were determined ot be 26.79 mg/L and >100 mg/L, respectively.

Executive summary:

In a study according to OECD TG 201, the effect of the test item on the growth of a unicellular green algal species Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) was determined. Exponentially growing cultures were exposed to various concentrations of the test item under static conditions. The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and thus, over several algal generations. Based on the results of a Range-Finding Test the following six test concentrations in a geometric series (with a spacing factor of 2.5) were tested: 1.024, 2.56, 6.4, 16, 40 and 100 mg/L (nominal concentrations). Untreated control ran parallel in the test. The measured concentration of the test item in the test solution was in the range of 95 – 105 % at the start and 93 – 98 % at the end of the test. There is evidence that the concentration of the tested substance was maintained within ± 20 % of the nominal concentration throughout the test. Therefore the analysis of the results was based on the nominal concentration values. The test item had a statistically significant inhibitory effect on the growth of Raphidocelis subcapitata after the exposure period of 72 hours based on the average specific growth rate and yield at the concentrations of 6.4, 16, 40 and 100 mg/L (Dunnett’s Test, 2 tailed, α=0.05) when compared to the control. The 72-h EC50 based on the growth rate was determined to > 100 mg/L and based on the yield the 72-h EC50 was determined to be 69.91 mg/L. The 72-h EC10 based on the growth rate was determined to be 26.79 mg/L and based on the yield the 72-h EC10 was determined to be 2.97 mg/L. Accordingly, the 72-h NOEC based on growth rate and yield was determined to be 2.56 mg/L and the LOEC as 6.4 mg/L.

Description of key information

In a 72-h algal growth inhibition test with Raphidocelis subcapitata, the obtained results showed that the test item had significant toxic effects on the growth of Raphidocelis subcapitata. The 72-hour NOEC based on growth rate and yield was determined to be 2.56 mg/L and the LOEC as 6.4 mg/L. The EC10 and EC50 based on growth rate were determined ot be 26.79 mg/L and >100 mg/L, respectively.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

26.79 mg/L

Additional information

In a study according to OECD TG 201, the effect of the test item on the growth of a unicellular green algal species Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) was determined. Exponentially growing cultures were exposed to various concentrations of the test item under static conditions. The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and thus, over several algal generations. Based on the results of a Range-Finding Test the following six test concentrations in a geometric series (with a spacing factor of 2.5) were tested: 1.024, 2.56, 6.4, 16, 40 and 100 mg/L (nominal concentrations). Untreated control ran parallel in the test. The measured concentration of the test item in the test solution was in the range of 95 – 105 % at the start and 93 – 98 % at the end of the test. There is evidence that the concentration of the tested substance was maintained within ± 20 % of the nominal concentration throughout the test. Therefore the analysis of the results was based on the nominal concentration values. The test item had a statistically significant inhibitory effect on the growth of Raphidocelis subcapitata after the exposure period of 72 hours based on the average specific growth rate and yield at the concentrations of 6.4, 16, 40 and 100 mg/L (Dunnett’s Test, 2 tailed, α=0.05) when compared to the control. The 72-h EC50 based on the growth rate was determined to > 100 mg/L and based on the yield the 72-h EC50 was determined to be 69.91 mg/L. The 72-h EC10 based on the growth rate was determined to be 26.79 mg/L and based on the yield the 72 -h EC10 was determined to be 2.97 mg/L. Accordingly, the 72-hour NOEC based on growth rate and yield was determined to be 2.56 mg/L and the LOEC as 6.4 mg/L.