L-Glutamic acid, N-(15-carboxy-1-oxopentadecyl)-, 5-(2,5-dioxo-1-pyrrolidinyl) ester

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009.12.01-2009.12.03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed in accordance with OECD principles of GLP with no deviations. Furthermore, the study fulfilled the validity criteria described in the OECD guideline.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))

Deviations:

no

Principles of method if other than guideline:

NA

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

PC2414

IUPAC Name:

PC2414

Details on test material:

- Name of test material (as cited in study report): PC2414
- Lot/batch No.: 0932L004
- Expiration date of the lot/batch: 2011-03-01

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
NA

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

In order to verify the tested concentrations, duplicate samples of each test concentration were taken at the initiation of the test, every 24 hours and at the termination of the test. Samples of approx. 40 mL were collected in 50-mL glass bottles and stored at -20 ± 2°C. Both samples with algae and without algae were taken. Samples from all test concentrations were analysed.

Test solutions

Vehicle:

no

Details on test solutions:

PC2414 was tested in the following nominal concentrations: 0 (control); 25; 50; 100; 200 and 400 mg/L. The concentrations were chosen in such a way that the lowest concentration should cause no effect and the highest 100% immobilization. Exposure concentrations of PC2414 should be maximised by keeping duration of the media preparation stage to a minimum according to the OECD guidance document on aquatic toxicity testing of difficult substances and mixtures. However, PC2414 was expected to hydrolyse rapidly and therefore, the test was mainly testing the toxicity of the hydrolysis products (NHS and impurity No.4).

A stock solution of 400 mg/L was prepared by weighing out 400 mg of PC2414 and mixing it in 1 L of test medium. Ultrasonication was used to achieve rapid dissolution of PC2414. The pH of the stock solution was adjusted to 7.8 ± 2 with NaOH. The test solutions were prepared by diluting the stock solution in the test medium. The pH in the test solutions was measured to be 7.6-7.9 and no further adjustment was necessary.

Freshly produced ISO-medium was used in the test. The medium was prepared from Milli-Q water, and salts were added to the water according to standard procedure. The pH was 7.6 and hardness of 250 ± 25 mg/L expressed as CaCO3.

The reference compound K2Cr2O7 was tested in the following concentrations: 0 (control); 0.2; 0.4; 0.7; 1.0; 1.4; and 2.0 mg/L..

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

A strain of Daphnia magna Straus (Cladocera, Crustacea), collected in Langedam, Birkerød, has been cultured at the testing laboratory used (DHI) since 1979. The culture is fed with Pseudokirchneriella subcapitata three times a day by a peristaltic pump system. Young animals (less than 24 hours old) from this culture were exposed to a dilution series of PC2414.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

The test was conducted with a semi-static test design.

Post exposure observation period:

NA

Test conditions

Hardness:

250 ± 25 mg/L expressed as CaCO3.

Test temperature:

The test was run in a climate room at 19.6 ± 0.6°C, in total darkness. The temperature was recorded continuously during the test by means of a thermologger.

pH:

Control group: 7.6 (start), 6.8 (end)
Higest concentration group: 7.9 (start), 7.3 (end)

Dissolved oxygen:

Control group: 99% (start), 99% (end)
Higest concentration group: 95% (start), 98% (end)

Salinity:

NA

Nominal and measured concentrations:

The results of the chemical analysis of the main hydrolysis product (Imp. No. 4) of PC2414 showed an average recovery of 97-102% of the initial concentration of hydrolysis product. The amount of hydrolysis product is shown to be stable throughout the test. The chemical analysis showed that the amount of hydrolysis product was 66 to 75% of the nominal test concentration of PC2414 (Table 1).

Details on test conditions:

The test was run as a semi-static test in 50-mL glass beakers with 25 mL of test solution. 20 animals were exposed at each concentration. 5 animals less than 24 hours old were transferred to each of 4 test beakers per concentration by use of a nylon net. The control group consisted of 30 animals, 6 beakers each containing 5 animals. After 24 hours of exposure, all test solutions were renewed. The number of immobile animals was recorded after 24 hours and after 48 hours. On this basis, the effect concentrations (EC) were determined. Dissolved oxygen and pH were measured at 0 hours, at 24 hours before and after renewal of test solutions and at 48 hours.

Reference substance (positive control):

yes

Remarks:

Analytical grade potassium dichromate (Sigma-Aldrich 31255, CAS No. 7778-50-9, Lot No. 62420)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 400 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

> 400 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

No effect was observed on the immobility of Daphnia magna after 48 hours in the control or in any of the tested concentrations and hence the 48 h EC10 and EC50 was >400 mg PC2414/L. (Table 2).

Results with reference substance (positive control):

Effect was observed on the immobility of Daphnia magna after 48 hours, thereby validating the study desing.The EC50 of the postivie control potassium dichromate (K2Cr2O7) was 0.62 (95% confidence interval: 0.51- 0.73) mg/L.

Reported statistics and error estimates:

The EC values for the reference substance K2Cr2O7 were calculated by use of the standard procedure Probit Analysis. As no effect of PC2414 and hydrolysis product was observed no effect concentrations were calculated.

Any other information on results incl. tables

Table 1. Analytical test results.

Nominal test concentration	t=0		t=24 old		t=24 new		t=48 old		Average recoveryin per cent of the initial concentration
	Hydrolysis product content in the test solutions	Recovery in per cent of the initial concentration	Hydrolysis product content in the test solutions	Recovery in per cent of the initial concentration	Hydrolysis product content in the test solutions	Recovery in per cent of the initial concentration	Hydrolysis product content in the test solutions	Recovery in per cent of the initial concentration
mg/L	(mg Imp. No. 4/L)	%	(mg Imp. No. 4/L)	%	(mg Imp. No. 4/L)	%	(mg Imp. No. 4/L)	%	%
200	141.1	100	140.4	100	142.3	101	150.7	107	102
400	289.2	100	288.4	100	278.9	96	265.4	92	97

Table 2. Test results

Testproduct/substance	Endpoint	Immobility (mg/L)
PC2414 and hydrolysis products	EC10	>400 mg/L
PC2414 and hydrolysis products	EC50	>400 mg/L
Potassium dichromate (K2Cr2O7)	EC50	0.62 (0.51- 0.73)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

There were no acute effect of PC2414 after 48 hours in the immobilization test using Daphnia magna , hence the 48 hour EC10 and EC50 was >400 mg PC2414/L.

Executive summary:

PC2414 was tested for acute toxic effects on the crustacean Daphnia magna according to the OECD Guideline 202 which is further specified in the ISO International Standard 6341. The test was carried out in compliance with the OECD Principles of Good Laboratory Practice (GLP). PC2414 was tested at the following nominal concentrations: 0 (control); 25; 50; 100; 200 and 400 mg/L. PC2414 is rapidly hydrolysed when mixed in water and therefore, the test was mainly testing the toxicity of the hydrolysis products (NHS and Impurity No. 4).

No effect was observed on the immobility of Daphnia magna after 48 hours in the control or in any of the tested concentrations, hence the 48 hour EC10 and EC50 was >400 mg PC2414/L.