(2S)-2-amino-3-(4-{[bis(sodiooxy)phosphoryl]oxy}phenyl)propanoic acid

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-06-04 to 2020-09-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

adopted April 13, 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

June 6, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: The samples were taken from the biological phase of the study. Duplicate samples from the freshly prepared test medium of the only test concentration and the control 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 concentration during the test period, duplicate samples from the test medium of the only test concentration and the control were collected at the end of the test (after 48 hours). All samples were stored in a freezer {< - 20 °C), protected from light, until analysis was performed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test medium of the only test item concentration of nominal 100 mg/L was prepared by dissolving 50.0 mg test item into 500 mL test water by intense stirring for 20 minutes. The test media was prepared just before introduction of the daphnids (= start of the test).

- Control: In the control, test water was used without addition of the test item.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain/clone: Daphnia magna (Straus), clone 5
- Justification for species other than prescribed by test guideline: Daphnia magna is the preferred species in accordance with the test guideline
- Age at study initiation: from 1.00 to 23.00 hours old
- Breeding conditions: The daphnids were bred in the laboratories under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in Elendt M4 medium. The test organisms were not first brood progeny. The daphnids in the stock culture were fed at least on all working days with green algae (Desmodesmus subspicatus).
- Source: the laboratories of ibacon

ACCLIMATION
- Acclimation period: not necessary, since the test was performed in the same medium as the culturing

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

2.5 mmol/L (= 250 mg/L) as CaCO3

Test temperature:

19.9 to 20.0 °C at test start;
20.2 °C at test end

pH:

7.6 to 8.3 at test start; 7.9 to 8.0 at test end; and thus the pH-value did not vary by more than 1.5 units

Dissolved oxygen:

9.0 to 9.1 mg/L at test start;
8.3 to 8.6 mg/L at test end

Nominal and measured concentrations:

Nominal concentrations: 100 mg/L;
Measured concentration: ranged from 49.669 to 52.139 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beakers of 100 mL volume containing approximately 60 mL of test medium
- Type: closed (The test vessels were covered with a lid to reduce the loss of water due to evaporation and to avoid the entry of dust into the solutions).
- No. of organisms per vessel: 5 animals
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Water (Elendt "M4")

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light: 8 h dark
- Light intensity: The light intensity was 520 to 760 lux (measured once during the test)

EFFECT PARAMETERS MEASURED: The number of immobilized daphnids were recorded after 24 and 48 hours.

RANGE-FINDING STUDY
- Test concentrations: 100 mg test item/L and a control
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Validity Criteria of the Study:
Control Immobilisation Rate was 0% and furthermore no daphnid showed signs of disease or stress; thus the validity criterion was met.
Dissolved Oxygen Concentration was ≥ 8.3 mg O2/L in in all treatment groups at the end of the test; thus validity criterion was met.

Biological Results:
Signs of Intoxication after 48 Hours: After 48 hours of exposure no immobilisation of the test animals was observed in the control and in the only test concentration of nominal 100 mg test item/L

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: 24-h EC50: 1.163 mg/L; 48-h EC50: 0.986 mg/L

Table 1: Summary of Biological Results

 

Nominal Concentration [mg test item/L]	% of immobilised daphnids after
	24 hours	48 hours
Control	0	0
100	0	0
EC50 [mg test item/L]	>100	>100
95% CI [mg test item/L]	n.d.	n.d.
NOEC [mgtest item/L]	100.0	100.0
LOEC [mg test item/L]	>100	>100

Values refer to nominal test concentrations

CI: Confidence interval

n.d.: not determinable

EC₅₀ Values, NOEC and LOEC were determined directly from the raw data.

 

Table 2: Summary of Analytical Results

Sample description Nominal [mg test item/L]	% of nominal1	RSD [%]	n
Control	n.a.	n.a.	4
100	102	2	4

¹ mean value of all measured samples per treatment group

RSD: relative standard deviation per treatment group

n: number of analysed samples

n.a.: not applicable

 

Table 3: Influence of the test item on the Mobility of Daphnia magna

 

Nominal Concentration [mg test item/L]	No. of Daphnia tested	No. of Immobilized Daphnia after		% of immobilized Daphnia after		Observations
		24 h	48 h	24 h	48 h	24 h	48 h
Control	5	0	0	0	0	0	0
	5	0	0			0	0
	5	0	0			0	0
	5	0	0			0	0
100	5	0	0	0	0	0	0
	5	0	0			0	0
	5	0	0			0	0
	5	0	0			0	0

Abbreviations for observations:

0: No abnormalities

 

Table 4: Results of the Analytical Determination of the Test Item in the Test Samples

 

Sample Description			Concentration found		Concentration calculated [mg test item/L]	Concentration nominal [mg test item/L]	% of nominal1
	Sampling Age
[mg test item/L]	day	[h]	[mg test item/L]	D.F.
Control	0	0	<LOD	2	n.a	0.000	n.a
Control	0	0	<LOD	2	n.a	0.000	n.a
Control	2	48	<LOD	2	n.a	0.000	n.a
Control	2	48	<LOD	2	n.a	0.000	n.a
100	0	0	52.139	2	n.a	100.000	104
100	0	0	49.669	2	n.a	100.000	99
100	2	48	51.113	2	n.a	100.000	102
100	2	48	50.368	2	n.a	100.000	101
						mean value (n=4):	102
						RSD (n=4):	2

 

¹ The tabulated results represent rounded results calculated on the exact raw data

LOD: Limit of Detection = 7.5 mg test item/L

n.a.: not applicable; RSD: Relative Standard Deviation; D.F.: Dilution factor

Validity criteria fulfilled:

yes

Conclusions:

In the 48-h acute toxicity of the test item on Daphnia magna according to OECD T 202, no immobilisation was observed in the control and in the 100 mg/L concentration group. In both groups no abnormal behaviour or appearance of test animals was noted. The 48-h NOEC was determined to be 100 mg/L. The 48-h LOEC and the EC50 values were determined to be > 100 mg/L. All validity criteria were met. Therefore, the study was considered as valid. Results are based on the nominal concentration.

Executive summary:

The influence of the test item on Daphnia magna was assessed in a 48-h Acute Immobilisation Test according to OECD TG 202. For this purpose, young Daphnia (< 24 hours old) were exposed to aqueous test media containing the test item for 48 hours. Based on results obtained from a static non-GLP preliminary experiment where the test item was previously shown to be stable in the test medium for 48 hours, daphnids were exposed to a single concentration of 100 mg/L (limit concentration). This limit-test was performed in compliance with the test guidelines in order to demonstrate that the test item has no toxic effects on the daphnids up to this concentration. The test method and the test species Daphnia magna are recommended by the test guidelines. This study encompassed two treatment groups (one test item concentration at nominal 100 mg/L and one control) each containing 20 individuals. The purpose of the analytical part of this study was to verify the concentration of the test item in the test medium. The analytically measured test item concentration remained within ± 20 % of the nominal during the test period of 48 hours; therefore, biological results are based on the nominal concentration. The test item concentration of 100 mg/L and the control (reconstituted water) were tested using 20 daphnids (divided into 4 concurrent subgroups) each. The mobility of the daphnids was determined in a static 48-hour test by visual observation after 24 and 48 hours. Environmental conditions were recorded. All measured values remained within acceptable ranges. No immobilisation or any abnormal behaviour was observed in the control and the 100 mg/L concentration group after 48 hours of exposure. All validity criteria were within acceptable limits and therefore the study was considered valid. The 48-h NOEC was determined to be 100 mg/L. The 48-h LOEC and the EC₅₀ values were determined to be > 100 mg/L.

Description of key information

In the 48-h acute toxicity of the test item on Daphnia magna according to OECD T 202, no immobilisation observed in the control and in the 100 mg/L concentration group. In both groups no abnormal behaviour or appearance of test animals was noted. The 48-h NOEC was determined to be 100 mg/L. The 48-h LOEC and the EC₅₀ values were determined to be > 100 mg/L. All validity criteria were met. Therefore, the study was considered as valid. Results are based on the nominal concentration (reference 6.1.3 -1).

Additional information

The influence of the test item on Daphnia magna was assessed in a 48-h Acute Immobilisation Test according to OECD TG 202. For this purpose, young Daphnia (< 24 hours old) were exposed to aqueous test media containing the test item for 48 hours. Based on results obtained from a static non-GLP preliminary experiment where the test item was previously shown to be stable in the test medium for 48 hours, daphnids were exposed to a single concentration of 100 mg/L (limit concentration). This limit-test was performed in compliance with the test guidelines in order to demonstrate that the test item has no toxic effects on the daphnids up to this concentration. The test method and the test species Daphnia magna are recommended by the test guidelines. This study encompassed two treatment groups (one test item concentration at nominal 100 mg/L and one control) each containing 20 individuals. The purpose of the analytical part of this study was to verify the concentration of the test item in the test medium. The analytically measured test item concentration remained within ± 20 % of the nominal during the test period of 48 hours; therefore, biological results are based on the nominal concentration. The test item concentration of 100 mg/L and the control (reconstituted water) were tested using 20 daphnids (divided into 4 concurrent subgroups) each. The mobility of the daphnids was determined in a static 48-hour test by visual observation after 24 and 48 hours. Environmental conditions were recorded. All measured values remained within acceptable ranges. No immobilisation or any abnormal behaviour was observed in the control and the 100 mg/L concentration group after 48 hours of exposure. All validity criteria were within acceptable limits and therefore the study was considered valid. The 48-h NOEC was determined to be 100 mg/L. The 48-h LOEC and the EC₅₀ values were determined to be > 100 mg/L.