diethyl pyridine-2,4-dicarboxylate

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:

21-Jun-2010 to 16-Jul-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

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:

2010

Report date:

2010

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:

other: Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), C.

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Mexoryl SBU

Cas Number:

41438-38-4

Molecular formula:

C11H13N04

IUPAC Name:

Mexoryl SBU

Constituent 2

Reference substance name:

diethyl pyridine-2,4-dicarboxylate

EC Number:

680-341-5

Cas Number:

41438-38-4

Molecular formula:

C11H13N04

IUPAC Name:

diethyl pyridine-2,4-dicarboxylate

Details on test material:

- Name of test material: MEXORYL SBU

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):not applicable

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

For the determination of the actual test item concentrations, duplicate samples were taken from each treatment at the start and end of each test medium renewal period.For sampling from the aged test media, the contents of the respective replicates were combined prior to sampling. All samples were stored deep-frozen (at about -20 °C) immediately after sampling. Based on pre-experiments for investigation of the storage stability (non-GLP), the test item proved to be stable in the test water under these storage conditions.The concentrations of the test item Mexoryl SBU were analyzed in the duplicate test media samples from the nominal concentrations of 46 and 100 mg/L from both sampling times (0 and 48 hours).The samples from the media of the lower test concentrations were not analyzed since the concentrations were below the 48 hour NOEC determined in this test and, therefore, not relevant for the interpretation of the biological test results. From the control, only one of the duplicate samples was analyzed per sampling time.The analytical procedure and results are described in the Attachment I -Analytical investigations.

Test solutions

Vehicle:

no

Details on test solutions:

At the start of each test medium renewal period, the test medium of the highest nominal concentration of 100 mg/L was prepared by completely dissolving 70.07 and 70.04 mg of test item, respectively, in 700 mL of test water using ultrasonic treatment for 15 minutes and intense stirring for 15 minutes at room temperature. Adequate volumes of these test media were diluted with test water to prepare the test media with the lower test item concentrations.The test media were prepared just before introduction of the daphnids (i.e., prior to the start of each test medium renewal period).

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

The study was performed with young daphnids of the species Daphnia magna Straus. A clone of this species (defined by the supplier as clone 5) was originally supplied by the University of Sheffield / UK in 1992. Since that time, the clone has been bred at Harlan Laboratories in reconstituted water of the quality identical to the water quality used in the tests (in respect to pH, main ions, and total hardness) and under temperature and light conditions identical to those of the tests.During breeding, daphnids are generally fed three times a week with an algal suspension of the green algae Desmodesmus subspicatus CHODAT, Strain No. 86.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany) and cultivated at Harlan Laboratories under standardized conditions or a mixture of this algal suspension and a commercial fish diet (Tetra Min® Hauptfutter, supplied by TETRA-Werke, 49304 Melle / Germany).At the start of the test, the organisms used in the test were 6 to 24 hours old and were not first brood progeny.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

not applicable

Test conditions

Hardness:

2.5 mmol/L

Test temperature:

The water temperature was 21 °C during the test.

pH:

At the beginning and end of the test medium renewal periods, the pH values of the test media were between 7.8 and 8.0 (for further details please see attached Table 2).

Dissolved oxygen:

The dissolved oxygen concentrations in the test media and control were at least 8.2 mg/L (for further details please see attached Table 3).

Salinity:

according to OECD medium

Nominal and measured concentrations:

The selection of the test concentrations was based on the results of the first test performed. The first test was performed as a limit test and significant immobilization was observed after 24 hours of test duration. Therefore, the test was repeated as a full test and the following nominal concentrations of MEXORYL SBU were tested: 4.6, 10, 22, 46 and 100 mg/L. Additionally, a control (test water without test item) was tested in parallel. Nominal concentrations above 100 mg/L were not tested in accordance with the test guidelines.The measured concentrations of the test item in the test media were between 93 and 96% of the nominal values at the start of the test medium renewal periods. Thus, the correct dosing of the test item was confirmed. At the end of the test medium renewal periods, 83 to 87% of the nominal values were found. Consequently, the test water renewal ensured nearly constant concentrations of the test item during the test and the biological results were related to the nominal test concentrations.

Details on test conditions:

Test WaterReconstituted test water according to ISO 6341 was used in the study. For further information please see section "Any other information on materials and methods incl. tables".The test water was aerated prior to the start of the study until oxygen saturation was reached. During the test period, the test water was not aerated.The test was performed in 100 mL glass beakers filled with 50 mL of test medium. The test vessels were covered with glass plates to reduce the loss of water by evaporation and to avoid the entry of dust into the solutions.The test vessels were labeled with the study number and all necessary additional information to ensure unique identification.The test was performed in a temperature-controlled room with continuous monitoring of the room temperature. The water temperature was maintained at 21 °C.A 16-hour light to 8-hour dark cycle with a 30-minute transition period was used. Light intensity during the light period was approximately between 520 and 680 Lux.The daphnids were not fed during the test.A semi-static test with test medium renewal after 24 hours was performed to keep the concentrations of the test item in the test media as constant as possible during the test period of 48 hours. After 24 hours, the test organisms were placed in clean test vessels with freshly prepared test medium of the corresponding concentration.

Reference substance (positive control):

yes

Remarks:

tested twice a year

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

The measured concentrations of the test item in the test media were between 93 and 96% of the nominal values at the start of the test medium renewal periods (for further information please see Attachment I - Analytical investigations). Thus, the correct dosing of the test item was confirmed. At the end of the test medium renewal periods, 83 to 87% of the nominal values were found. Consequently, the test water renewal ensured nearly constant concentrations of the test item during the test and the biological results were related to the nominal test concentrations.During the first 24 hours of the test, no immobilized test organisms were determined in the control and up to and including the test item concentration of 46 mg/L. At the highest test concentration of 100 mg/L, the immobilization was 10%. After 48 hours of exposure, the immobilization rate had not increased. Although, this low immobilization rate of 10% is tolerated by the guidelines in the control, it is estimated as a toxic effect of the test item, taking into account also the results of the first main test, performed as a limit test, with significant mortality after 24 hours.The 24- and 48-hour EC50 and EC100 were >100 mg/L.No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the entire test duration.At the beginning and end of the test medium renewal periods, the pH values of the test media were between 7.8 and 8.0. The dissolved oxygen concentrations in the test media and control were at least 8.2 mg/L , and the water temperature was 21 °C during the test.The test is considered to be valid, as in the control, no daphnids showed immobilization or other signs of disease or stress (e.g., discoloration or unusual behavior such as trapping at the surface water). Furthermore, the dissolved oxygen concentration at the end of the test was ≥3 mg/L in the control and test vessels.

Results with reference substance (positive control):

For evaluation of the quality of the daphnia clone and the experimental conditions, potassium dichromate is tested as a positive control twice a year. The result of the latest positive control test of 1st April 2010 (48-hour EC50: 0.43 mg/L, study C86933) proved the sensitivity of the test organisms and the validity of the test design (internal historical range of 48-hour EC50 from 2000 to 2010: 0.43 to 1.1 mg/L).

Reported statistics and error estimates:

The NOEC, EC0 and EC100 were determined directly from the raw data.The 24- and 48-hour EC50 could not be calculated since none of the responses exceeded 50%. The EC50 was, therefore, also determined directly from the raw data.

Any other information on results incl. tables

The measured concentrations of the test item in the test media were between 93 and 96% of the nominal values at the start of the test medium renewal periods(for further information on analytical results see Attachment I - Analytical Investigations). Thus, the correct dosing of the test item was confirmed. At the end of the test medium renewal periods, 83 to 87% of the nominal values were found.Consequently, the test water renewal ensured nearly constant concentrations of the test item during the test and the biological results were related to the nominal test concentrations.

During the first 24 hours of the test, no immobilized test organisms were determined in the control and up to and including the test item concentration of 46 mg/L. At the highest test concentration of 100 mg/L, the immobilization was 10%. After 48 hours of exposure, the immobilization rate had not increased. Although, this low immobilization rate of 10% is tolerated by the guidelines in the control, it is estimated as a toxic effect of the test item, taking into account also the results of the first main test, performed as a limit test, with significant mortality after 24 hours.

 

The 24- and 48-hour EC₅₀ and EC₁₀₀ were>100 mg/L.

 

No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the entire test duration.

 

At the beginning and end of the test medium renewal periods, the pH values of the test media were between 7.8 and 8.0 (for further information see atteched Table 2). The dissolved oxygen concentrations in the test media and control were at least 8.2 mg/L (for further information see attached Table 3) , and the water temperature was 21°C during the test.

 

The test is considered to be valid, as in thecontrol, no daphnids showed immobilization or other signs of disease or stress (e.g., discoloration or unusual behaviour such as trapping at the surface water). Further more, the dissolved oxygen concentration at the end of the test was ≥3 mg/L in the control and test vessels.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Executive summary:

The acute toxicity of the test item MEXORYL SBU to Daphnia magna was determined in a 48 hour semi-static test according to the EU Commission Directive 92/69/EEC, Part C.2, the Commission Regulation (EC) No 440/2008, Part C.2 and the OECD Guideline for Testing of Chemicals, No. 202 (2004).

 

The nominal test item concentrations tested were 4.6, 10, 22, 46 and 100 mg/L. Additionally, a control group was tested in parallel.

 

The measured concentrations of the test item in the test media were between 93 and 96% of the nominal values at the start of the test medium renewal periods. Thus, the correct dosing of the test item was confirmed. At the end of the test medium renewal periods, 83 to 87% of the nominal values were found.Consequently, the test water renewal ensured nearly constant concentrations of the test item during the test and the biological results were related to the nominal test concentrations.

 

The biological test results (based on nominal test item concentrations) were as follows:

 

– 24-hour EC50:	>100	mg/L
– 24-hour EC0:	46	mg/L
– 24-hour EC100:	>100	mg/L
– 48-hour EC50:	>100	mg/L
– 48-hour EC0and
48-hour NOEC:	46	mg/L
– 48-hour EC100:	>100	mg/L