(4R)-2-oxo-1,3-oxazolidine-4-carboxylic 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:

Experimental start date: November 01, 2019; Experimental completion date: December 19, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 2006, corrected 2011

Deviations:

no

Remarks:

96 h exposure duration

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All test concentration groups and the control
- Sampling method: For the determination of the test item concentrations, duplicate samples (10 mL) were taken from the test media of all test concentration groups and the control at the start and at the end of the test (96 hours). For sampling at the end of the test, the test medium of the treatment replicates was pooled.
- Sample storage conditions before analysis: All samples were stored frozen (-20 ± 5 °C) immediately after sampling until analysis.
- Analysed samples: The concentrations of the test item were measured in one of the duplicates for all taken samples.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: At the start of the test, the test medium of the highest nominal concentration of 100 mg/L was prepared by mixing 100.20 mg of the test item into 1000 mL test water using intense stirring for 15 minutes at room temperature. The test medium of the highest test concentration was diluted with test water to prepare the test media of the lower test concentrations
- Controls: A control was tested in parallel (test water without test item). The test design included three replicates per test concentration and six replicates of the control.
- Evidence of undissolved material: No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the entire test duration. The test media were prepared just before the start of the exposure.

Test organisms (species):

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

Details on test organisms:

•TEST ORGANISM
- Common name: Green algae
- Strain: No. 61.81 SAG
- Source: Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany
- Age of inoculum (at test initiation): An inoculum culture of algae was set up three days before the start of the test.
- Method of cultivation: The algae were cultivated at IES Ltd Laboratories under standardized conditions according to the test guidelines.

•ACCLIMATION
- Acclimation period: The algae were cultivated under the test conditions and kept in the exponential growth phase until inoculation of the test solutions. The inoculum culture was incubated under the same conditions and dilution media as the test cultures. After the end of the evaluations, the algae cultures in the treatments including the control were disposed and not be used for any further testing.
- Culturing media and conditions: Same as test

Test type:

static

Water media type:

freshwater

Remarks:

Reconstituted test water (AAP Medium) prepared according to the test guideline was used for algal cultivation and testing. Analytical grade salts were dissolved in sterile purified water.

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

In agreement with the Test Guideline (TG) the normal exposure duration of 72 h was extended to 96 h because all validity criteria of in paragraph 11 of the TG could be met.

Hardness:

The calculated water hardness of the test water was 0.15 mmol/L (= 15 mg/L as CaCO3).

Test temperature:

The temperature during the test was maintained at 21 °C.

pH:

- The pH in the test water (AAP medium) was 7.5.
- The pH of the test media was in the range of 6.5 to 8.2 during the test period.
- The pH rose in the control from 7.5 at test start to 8.1 at test end fulfilling the requirement of the OECD guideline that the pH of the control medium should not increase by more than 1.5 units during the test.

Nominal and measured concentrations:

- Nominal test item concentrations were 4.6, 10, 22, 46 and 100 mg/L.
- Determined test item concentrations
• in the fresh solutions (day 0, 0 h) 4.71, 10.8, 22.4, 49.0 and 109 mg/L corresponding to 102, 108, 102, 106 and 109 % of nominal (respectively) and
• in the aged solutions (day 4, 96 h) 4.97, 11.0, 23.5, 48.7 and 111 mg/L corresponding to 108, 110, 107, 106 and 111 % of nominal (respectively).
• The measured concentrations of in the test media of the test concentrations of 4.6 to 100 mg/L were between 102 and 109 % of the nominal values at the start of the test and between 106 and 111 % at the end of the test. Thus, the correct dosing of the test item was confirmed and the test item was stable in the test media over the test period of 96 hours. Therefore, the endpoint values were reported on the nominal concentrations of the test item.

Details on test conditions:

•DETAILS ON TEST CONDITIONS
•TEST SYSTEM
- Test vessel: Erlenmeyer flasks. Each test flask was loosely covered with a glass lid, thereby adequate CO2 be transferred from surrounding air to the test solution.
- Material, size, headspace, fill volume: Glass, 75 mL, not firmly closed, 30 mL
- Aeration: No
- Initial cells density: 5000 algal cells/mL, corresponding to 0.729 ∙ 10^4 relative fluorescence units. The algal cell density in the pre-culture was determined using an electronic particle counter (Cell Counter CASY TT, OLS, Bremen/Germany).
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

•GROWTH MEDIUM
- Standard medium used: Yes, AAP-Medium

•TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Sterile purified (reconstituted) tap water
- Macro-nutrients:
•Ingredient / Concentration
• NaHCO3 / 15.0 mg/L
• K2HPO4 / 1.044 mg/L
• MgSO4 × 7 H2O / 14.6 mg/L
• MgCl2 × 6 H2O / 12.16 mg/L
• CaCl2 × 2 H2O / 4.41 mg/L
• NaNO3 / 25.5 mg/L
- Trace elements:
•Ingredient / Concentration
• H3BO3 / 186.0 µg/L
• MnCl2 × 4 H2O / 415.0 µg/L
• ZnCl2 / 3.27 µg/L
• CoCl2 × 6 H2O / 1.43 µg/L
• CuCl2 × 2 H2O / 0.012 µg/L
• Na2MoO4 × 2 H2O / 7.26 µg/L
• FeCl3 × 6 H2O / 160.0 µg/L
• Na2EDTA × 2 H2O: 300.0 µg/L

- Culture medium different from test medium: No

•OTHER TEST CONDITIONS
- Sterile test conditions: No
- Adjustment of pH: Since the pH of the test media at the concentrations of 22, 46 and 100 mg/L was 5.8, 3.9 and 3.4, respectively, after preparation and outside of the recommended range of 6 to 9, the pH of these test media was adjusted to 6.5 with 0.1 M sodium hydroxide solution.
- Photoperiod: Continuously illuminated
- Light intensity and quality: LED lights installed above the test flasks; The light intensity at the level of the test solutions was approximately 76 µE/sm² (range: 75 to 78), LED measured at nine places in the experimental area). The light intensity over the incubation area was within a ±15 %-deviation from the average light intensity as recommended by the guideline.

•EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Fluorimeter; A small volume (100 μL per sampling) of the algal suspension was withdrawn from each test flask at 24, 48, 72 and 96 hours after the start of the exposure, for the measurement of algal biomass, and was not replaced. The algal biomass in the samples was determined by fluorescence measurement (SpectraMax I3x, Molecular Devices Ltd, Wokingham Berkshire/UK). The measurements were performed at least in duplicate at an excitation of 440 nm and emission of 680 nm.
- Other: At the end of the test, a sample was taken from the control and from the nominal test concentration of 100 mg/L to determine a potential influence of the test item on the algal cells. The shape and size of the algal cells were examined microscopically.

•TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 2.2

•RANGE-FINDING STUDY
- Test concentrations: 0.1 / 1.0 / 10 / 100 mg/L
- Results used to determine the conditions for the definitive study:
•Concentration [mg/L] / Inhibition of Average Growth after 96 hours [%] / Inhibition of Yield after 96 hours [%]
0.10 / -0.8 / -4.8
1.0 / 0.2 / 1.2
10 / 1.4 / 7.7
100 / 2.3 / 12

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (CAS 7778-50-9), Batch No. MKBR3876V), obtained from Sigma-Aldrich

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

Remarks on result:

other:

Remarks:

95% CI n.d.; NOEC 22 mg/L (Williams t-test, one-sided smaller, α = 0.05); LOEC 46 mg/L.

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

Remarks on result:

not determinable

Remarks:

95% CI n.d.

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

71 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

yield

Remarks on result:

other:

Remarks:

95% CI 41- >100 mg/L (Probit analysis); NOEC 22 mg/L (Williams t-test, one-sided smaller, α = 0.05); LOEC 46 mg/L.

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

yield

Remarks on result:

not determinable

Remarks:

95% CI n.d.

Details on results:

- Exponential growth in the control: Yes
- Observation of abnormalities: The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal concentration of 100 mg/L and the algal cells in the control. The shape and size of the algal cells were not affected by the test item at any of the test concentrations tested.
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: 1.3 mg potassium dichromate/L with a 95 % confidence intervall of 1.2 to 1.4 mg/L for growth rate and 0.47 mg potassium dichromate/L with a 95 % confidence intervall of 0.44 to 0.49 for yield
- Other: IES Study 20190393 (Experimental Starting Date: November 01, 2019; Experimental Completion Date: November 08, 2019)

Reported statistics and error estimates:

AUC, growth rate and yield were calculated for each test flask. The mean values for AUC, growth rate and yield were calculated for each treatment. Only the 72 and 96-hour EC10 values for the inhibition of AUC and the 72-hour EC10 values for the inhibition of yield were calculated (Probit Analysis using linear maximum likelihood regression). All the others ECx values (72 and 96-hour EC10 ,EC20 and EC50 values) for the inhibition of AUC, average growth rate and yield and their 95 % confidence intervals could not be calculated because of the low inhibitory effect of the test item on the algal growth at the tested concentrations. For the determination of the LOEC and NOEC, the AUC, average growth rate and yield at the test concentrations were compared to the control values by Williams t-test. Statistical analysis was performed using ToxRat Professional®.

The test was valid since the following performance criteria (according to the OECD 201) were met.
- In the control, the biomass increased by a factor of 92 over 72 hours. (Criterion: increase by at least a factor of 16 within three days).
- The mean coefficient of variation of the daily growth rates in the control (section-bysection growth rates) during 72 and 96 hours was 19%. (Criterion: must not be higher than 35%).
- The coefficient of variation of the average specific growth rates in the replicates of the control after 72 and 96 hours was 1.0 and 1.2%, respectively. (Criterion: must not be higher than 7%).

The biological results of the study (based on nominal concentrations) are summarized in the table below (corresponds to Table 9 of the report):

Parameter	after 72 hours			after 96 hours
	AUC	Growth rate	Yield	AUC	Growth rate	Yield
	[mg/L]
EC10^a	42^a	>100^b	71^a	88^a	>100^b	>100^b
95% CI	28-57	n.d.	41 - >100	51- >100	n.d.	n.d.
EC20^a	>100^b	>100^b	>100^b	>100^b	>100^b	>100^b
95% CI	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
EC50^a	>100^b	>100^b	>100^b	>100^b	>100^b	>100^b
95% CI	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
NOEC^*	22	22	22	22	100	22
LOEC	46	46	46	46	>100	46

a: Probit Analysis

b: EC_X values of the test item could not be calculated because of the low inhibitory effect of the test item on the algal growth at the tested concentrations.

^*: Williams t‑test, one-sided smaller, α = 0.05

95% CI: 95% confidence interval

n.d.: Could not be determined

Validity criteria fulfilled:

yes

Conclusions:

The test item had no adverse effect on the growth rate of freshwater green algae (Raphidocelis subcapitata) over a test period of 96 hours up to nominal test concentrations of 100 mg/L.

Executive summary:

The acute toxicity of the test item on the growth of a freshwater green algal species (Raphidocelis subcapitata) was investigated in a 96-hour static test according to OECD TG 201, adopted 2006 (corrected 2011). The validity criteria were met. Based on the range-finding test, nominal test item concentrations were 4.6, 10, 22, 46 and 100 mg/L. A control (test water without test item) was tested in parallel. AAP-medium was used as test medium.

Duplicate samples (10 mL) were taken from the test media of all test concentration groups and the control at the start and at the end of the test (96 hours). The concentrations were measured with a HPLC method with MS detection using an external standard. The measured concentrations of in the test media of the test concentrations of were between 102 and 109% of the nominal values at the start of the test and between 106 and 111% at the end of the test. The endpoint values were reported on the nominal concentrations of the test item. Erlenmeyer flasks (glass, size 75 mL, fill volume 30 mL), loosely covered with a glass lid allowing adequate carbon dioxide transfer from surrounding air to the test solution, served as test vessels. In agreement with the Test Guideline the normal exposure duration of 72 h was extended to 96 h. The test item had no adverse effect on the growth rate of freshwater green algae (Raphidocelis subcapitata) and resulting 72-hour ErC10 and ErC50 values were >100 mg/L.

Description of key information

72-hour and 96-hour ErC50 >100 mg/L, freshwater green algae (Pseudokirchneriella subcapitata), OECD TG 201

72-hour and 96-hour ErC10 >100 mg/L, freshwater green algae (Pseudokirchneriella subcapitata), OECD TG 201

Additional information

The toxicity of the substance to a freshwater green algal species (Pseudokirchneriella subcapitata) was studied under GLP to OECD TG 201. No adverse effect on the growth rate of algae was observed over the exposure period of 72 hours, which was extended to 96 hours. A minimal effect on the biomass was seen at the highest test concentration. The 72-hour and 96-hour ErC10 and ErC50 values were greater than 100 mg/L.