Potassium (2S)-4-carboxy-2-(tetradecanoylamino)butanoate

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:

11 September 2012 to 13 September 2012

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese guidelines for studies on new chemical substance as required by the Law Concerning the Evaluation of Chemical Substances and Regulation of their Manufacture 1973, amended 2006 and 2009.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF 2-7-2-1 The guidelines related to the study reports for the registration application of pesticide (Ref. No. 12-Nousan-8147 on 24 November 2000)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Concentration of the test material in the test solutions was determined at the beginning and end of the renewal periods. The samples were analysed by an HPLC-MS method.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
An amount of 50 mg test substance was diluted in 500 mL ISO medium (stock solution concentration: 100.0 mg/L). The test solutions were prepared by appropriate dilution of this stock solution (see Table 1).
Stock solutions were prepared fresh for each renewal.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Freshwater water flea
- Strain: Daphnia magna
- Age at study initiation (mean and range, SD): less that 24 hours old
- Method of breeding:
- Feeding during test: Daphnia were not fed during the test

ACCLIMATION
- Acclimation period: There was no acclimatisation because the water used was similar to the culture water

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

None

Hardness:

The reconstituted water (ISO medium) had an approximate theoretical total hardness of 249 mg/L (as CaCO3).

Test temperature:

Test flask: 20.8 – 21.2 °C
Climatic chamber: 20.4 – 21.4 °C

pH:

The pH of the test solution was not adjusted and not varied by more than 1.5 units in any one test. The pH was measured at the start and at the end of the renewal periods in each test vessel and was in the range of 7.49 – 7.55.

Dissolved oxygen:

The dissolved oxygen concentration was measured in each test vessel at the start and at the end of the renewal periods and was in the range of 7.4 – 7.5 mg/L.

Salinity:

N/A

Nominal and measured concentrations:

Nominal: 6.25, 12.5, 25, 50 and 100 mg/L
Measured geometric mean: 4.5; 8.2; 13.3; 27.2 and 36.8 mg/L

Since the measured concentrations deviated more than 20 per cent from the nominal in all cases during the experiment, biological results are based on the measured geometric mean concentrations.

Details on test conditions:

TEST SYSTEM
- Renewal rate of test solution (frequency/flow rate): every 24 hours
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
Reconstituted water (ISO medium, according to OECD 202) was used as dilution water for both the range finding and definitive tests. The same composition of reconstituted water was used for the tests and for breeding the test animals.

OTHER TEST CONDITIONS
- Adjustment of pH: Not adjusted
- Photoperiod: 16 hour light and 8 hour dark cycle

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
The immobility or mortality of the Daphnia was determined by visual observation 24 and 48 hours after the start of the test. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker are considered to be immobile.
The number of immobilised animals and the percentage of immobility were determined at 24 and 48 hours.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: N/A

RANGE FINDING STUDY
A concentration range-finding test was conducted to determine the approximate toxicity of the test material so that appropriate test concentrations can be selected for use in the definitive test. Ten daphnids (divided into two replicates) in each test concentration and control were exposed for 48 hours under static and semi-static conditions in parallel. In the semi-static study the frequency of the water renewal periods was 24 hours.
During the formulation procedure the test solutions were prepared by the similar method described above.
The concentration levels used and results (48 h) of the preliminary range-finding tests are summarised in Tables 2 and 3.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate, last tested on 09-10 August 2012

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

56.48 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

34.64 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % confidence limits: 27.86 - 51.91 mg/L

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 36.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

13.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

27.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Under the conditions of this Daphnia magna acute immobilisation study, the calculated and observed endpoints for the effect of the test substance, were as follows:
The 24h EC50 value: 56.48 mg/L (95 % confidence limits could not be calculated)
The 48h EC50 value: 34.64 mg/L (95 % confidence limits: 27.86 – 51.91 mg/L)
The 48h EC100 value: > 36.8 mg/L
The 48h No-Observed Effect Concentration (NOEC): 13.3 mg/L
The 48h Lowest Observed Effect Concentration (LOEC): 27.2 mg/L

Results with reference substance (positive control):

Potassium dichromate, 24h EC50: 0.98 mg/L, (95 % confidence limits: 0.92 – 1.06 mg/L).

Reported statistics and error estimates:

The 24 and 48 hours EC50 values with 95 % confidence limits were calculated using Probit analysis by SPSS PC+ software. The 48 hours EC100 values were determined directly from the raw data.
For the determination of the LOEC and NOEC, the immobilisation at the test concentrations was tested on significant differences to the control values by Dunnett’s Test using TOXSTAT software.

Table 6: Immobilisation

Concentration		No. of treated animals	Immobilised animals
Nominal (mg/L)	Measured (mg/L)		24 hours		48 hours
			No.	%	No.	%
Control	0.0	20	0	0	0	0
6.25	4.5	20	0	0	0	0
12.5	8.2	20	0	0	0	0
25	13.3	20	0	0	2	10
50	27.2	20	2*	10	5*	25
100	36.8	20	4*	20	12*	60

* Statistically significantly different in comparison to the control values (Dunnett’s Test: α = 0.05)

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the test, the test material was determined to have an EC50 of 34.64 mg/L at 48 hour with 95 % confidence limits of 27.86 - 51.91 mg/L. The NOEC and LOEC were also determined to be 13.3 mg/L and 27.2 mg/L, respectively.

Executive summary:

The acute toxicity of the test material to invertebrates was determined using Daphnia magna in a GLP study performed in accordance with the standardised guidelines OECD 202, EPA OPPTS 850.1010, EU Method C.2, and Japanese guidelines on the testing of new chemical substances.

Twenty Daphnids per concentration were exposed to the test material over 48 hours under freshwater semi-static conditions, and observed for immobilisation at 24 and 48 hours. Animals were exposed at measured concentrations of 4.5, 8.2, 13.3, 27.2 and 36.8 mg/L, analysed using HPLC-MS.

A untreated control was run concurrently for comparison. Potassium dichromate was used as a reference control, and was last tested on 09 - 10 August 2012.

Under the conditions of the test, the test material was determined to have an EC50 of 34.64 mg/L at 48 hour with 95 % confidence limits of 27.86 - 51.91 mg/L. The NOEC and LOEC were also determined to be 13.3 mg/L and 27.2 mg/L, respectively.

Description of key information

EC50 = 34.64 mg/L at 48 hour (95 % confidence limits 27.86 - 51.91 mg/L), NOEC = 13.3 mg/L, LOEC 27.2 mg/L, OECD 202, EPA OPPTS 850.1010, EU Method C.2, and Japanese guidelines on the testing of new chemical substances, Sipos (2013b)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

34.64 mg/L

Additional information

In the key study (Sipos, 2013b) the acute toxicity of the test material to invertebrates was determined using Daphnia magna in a GLP study performed in accordance with the standardised guidelines OECD 202, EPA OPPTS 850.1010, EU Method C.2, and Japanese guidelines on the testing of new chemical substances.

Twenty Daphnids per concentration were exposed to the test material over 48 hours under freshwater semi-static conditions, and observed for immobilisation at 24 and 48 hours. Animals were exposed at measured concentrations of 4.5, 8.2, 13.3, 27.2 and 36.8 mg/L, analysed using HPLC-MS.

A untreated control was run concurrently for comparison. Potassium dichromate was used as a reference control, and was last tested on 09-10 August 2012.

Under the conditions of the test, the test material was determined to have an EC50 of 34.64 mg/L at 48 hour with 95 % confidence limits of 27.86 - 51.91 mg/L. The NOEC and LOEC were also determined to be 13.3 mg/L and 27.2 mg/L, respectively.