3-[(2-acetamidoacetyl)amino]propanoic acid

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:

January to February 2016

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted March 2006, corrected July 28, 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Comissin Regulation (EC) No 761/2009, C.3., 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Sampling:
Duplicate samples from the freshly prepared test media (containing algae) of all test concentrations and from the controls were taken at the start of the test.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of all test concentrations and the controls (containing algae) were taken at the end of the test (after the 72 hours test period) by pouring together the contents of the test beakers of each treatment.
Additional samples of the control blank were taken at test start and test end.
Analyses:
The concentrations of the active ingredient Acetyl glycyl ß-alanine in the test item GenoWhite were analysed in the duplicate test media samples from all test concentrations, from both sampling times (0 and 72 hours). One of the duplicate control samples was analysed at test start and duplicate control samples were analysed at test end (72 hours).

Vehicle:

yes

Remarks:

test item dissolved in test water

Details on test solutions:

Pre-Experiments: Pre-experiments were performed to determine a suitable concentration range and to establish suitable methods to prepare the test solutions.
Test Concentrations: 100, 45.5, 20.7, 9.4 and 4.3 mg test item/L, and a control.
Control: In the control, test water was used without addition of the test item.
Dosage of Test Item: The test medium of the highest test concentration of nominal 100 mg test item/L was prepared by dissolving 101.6 mg test item into 1016 mL test water by intense stirring for 15 minutes. Adequate volumes of this test medium were diluted with test water to prepare the test media of the other desired test concentrations.
The test media were prepared just before introduction of the algae (= start of the test).
Appearance of the Test Item in Test Medium:There were no remarkable observations.

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata (KORSHIKOV), Strain No. 61.81 SAG, formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV)
Origin: The algae were supplied by the Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen", 37073 Göttingen, Germany.
Breeding Conditions: The algae were cultivated in the laboratories of ibacon under standardised conditions according to the test guidelines.
Reference Item: For the evaluation of the quality of the algae and the experimental conditions the reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Test type:

static

Water media type:

freshwater

Remarks:

Reconstituted Water (OECD medium)

Limit test:

no

Total exposure duration:

72 h

Hardness:

Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L) as CaCO3

Test temperature:

The temperature was measured daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks.
Water temperature was: 22 to 23 °C

pH:

The pH was measured in all test item concentrations and the control at the start and the end of the test.
pH was: 8.1 at test start and 9.0 to 9.2 at test end

Nominal and measured concentrations:

At the start of the test 98% of the nominal test concentrations were found (average of all test concentrations). After 72 hours test duration, 99% of the nominal value was determined (average of all test concentrations). During the test the algae were exposed to a mean of 98% of nominal. Therefore, all reported results refer to nominal concentrations.

Details on test conditions:

Light Regime: Continuous illumination
Light Intensity:The light intensity was measured once during the test at 6 positions distributed over the experimental area at the surface of the test media.
Mean light intensity: 5517 lux (range: 5180 to 5730 lux)
Recording: Test conditions were recorded with suitable instruments and documented in the raw data.

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:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 100 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:

other: yield

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:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

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

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

LOEC

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:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

Validity Criteria of the Study

Cell Density Increase in Control Cultures:At least by a factor of 16 within 72 hours
142.5-fold increase within 72 hours and thus, the validity criterion was met.

Coefficient of Variation of Sectional (Daily) Growth Rates in Control Cultures: Must not exceed 35 %
25.4 % and thus, the validity criterion was met.

Coefficient of Variation of Average Growth between Control Replicates: Must not exceed 7 %
1.9 % and thus, the validity criterion was met.

Biological Results

Growth Inhibition:
The 72-hour EC50, the 72-hour EC20 and the 72-hour EC10 for the parameters growth rate and yield could not be calculated due to the lack of effects of the test item. Therefore the 72-hour EC50 and the 72-hour EC10 was determined to be higher than the 100 mg test item/L, the highest concentration tested.
The 72-hour NOEC was determined to be at least 100 mg test item/L for the parameters growth rate and yield and the associated 72-hour LOEC was determined to be higher than 100 mg test item/L for both parameters.

Microscopic Examination:
The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing up to a nominal test concentration of 100 mg test item/L and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to the highest concentration tested.

Reported statistics and error estimates:

Based on the calculated cell densities, the 72-hour ErC50 and the 72-hour EyC50 (see Definitions) could not be calculated by Probit analysis due to the lack of effects and were therefore determined directly from the raw data.
For the determination of the 72-hour LOEC and the 72-hour NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Dunnett’s t-test (yield and growth rate).
The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

Validity criteria fulfilled:

yes

Conclusions:

The influence of acetylglycyl beta-alanine on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static dose-response test. The 72-hour NOEyC and 72-hour NOErC was determined to be at least 100 mg test item/L. The NOEC for yield and growth rate might even be higher than this concentration, but concentrations in excess of nominal 100 mg test item/L or the limit of solubility have not been tested according to the recommendations of the test guidelines. The 72-hour LOEC and the 72-hour EC50 for yield and growth rate were clearly higher than 100 mg test item/L. These values could not be quantified due to the absence of toxicity of GenoWhite up to nominal 100 mg test item/L.

Executive summary:

The influence of acetylglycyl beta-alanine on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static dose-response test (OECD 201 "FreshwaterAlgae and Cyanobacteria, Growth Inhibition Test", 2009).

Test Species: Pseudokirchneriella subcapitata, Strain No. 61.81 SAG cultivated in the laboratories of ibacon; original source: "Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissen-schaften, Universität Göttingen", 37073 Göttingen, Germany.

Test Design: This study encompassed 6 treatment groups (5 dose rates of the test item and a control) with three replicates per test concentration and six replicates for the control. At test start 50 mL of the test concentrations were inoculated with 5000 algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72 hours for determination of cell densities by spectrophotometrical measurement.

Endpoints: Yield and growth rate of the algae

Test Concentrations: 100, 45.5, 20.7, 9.4 and 4.3 mg test item/L, and a control.

Test Conditions: Water temperature: 22 to 23 °C; pH values at test start 8.1, at the end of the test 9.0 to 9.2; continuous illumination; mean light intensity: 5517 lux (5180 to 5730 lux).

Table 1. Biological Results

Parameter                     Yield [mg test item/L]              Growth rate [mg test item/L]

72-hour EC50                     >100                                          >100

95 % conf. interval                 n.d.                                          n.d.

72-hour EC20                     >100                                          >100

95 % conf. interval                 n.d.                                          n.d.

72-hour EC10                     >100                                          >100

95 % conf. interval                 n.d.                                          n.d.

72-hour NOEC                   ≥100                                        ≥100

72-hour LOEC                     >100                                         >100

n.d. = not determinable Values refer to nominal test concentrations

Analytical Results:

The quantification of the active ingredient Acetyl glycyl ß-alanine in the test item GenoWhite was performed using liquid chromatography with UV detection.

At the start of the test 98% of the nominal test concentrations were found (average of all test concentrations). After 72 hours test duration, 99% of the nominal value was determined (average of all test concentrations). During the test the algae were exposed to a mean of 98% of nominal. Therefore, all reported results refer to nominal concentrations.

Conclusion:

The 72-hour NOEyC and 72-hour NOErC was determined to be at least 100 mg test item/L. The NOEC for yield and growth rate might even be higher than this concentration, but concentrations in excess of nominal 100 mg test item/L or the limit of solubility have not been tested according to the recommendations of the test guidelines. The 72-hour LOEC and the 72-hour EC50 for yield and growth rate were clearly higher than 100 mg test item/L. These values could not be quantified due to the absence of toxicity of GenoWhite up to nominal 100 mg test item/L.

Description of key information

Static dose response study: Toxycity to Pseudokirchneriella subcapitata in an Algal Growth Inhibition Test, OECD 201

NOEC (72 hour, growth rate) ≥100 mg test item/L

NOEC (72 hour, yield) ≥100 mg test item/L

LOEC (72 hour, growth rate) >100 mg test item/L

LOEC (72 hour, yield) >100 mg test item/L

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L