3-sec-[C15-18-(branched and linear)-alk-2-enyl]pyrrolidine-2,5-dione

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 March 2007 to 30 March 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

other: read-across target

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Water samples were taken from the control (replicates R1 - R4 pooled) and the 100 mg/L loading rate WAF test groups (replicates R1 - R2 and R3 - R4 pooled) at 0 and 48 hours for quantitative analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Due to the low aqueous solubility and complex nature of the test material for the purposes of the definitive test the test material was prepared as a Water Accommodated Fraction (WAF).
- An amount of test material (1000 mg) was added to the surface of 10 litres of reconstituted water to give the 100 mg/L loading rate. After the addition of the test material, the reconstituted water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. This was stirred for 23 hours. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. The WAF was removed by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 100 mg/L loading rate WAF. Microscopic observations of the WAF were performed after filtering and showed no micro-dispersions or particles of test material present.
- Preliminary recovery and stability analysis indicated the test material to be light sensitive and therefore, the test material was weighed out and diluted under a non-actinic light source and the test vessels and sample bottles were shielded from the light.
- The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 and 48 hours

Validation of Mixing Period
- Preliminary investigational work was carried out to determine whether stirring for a prolonged period produced significantly higher levels of total organic carbon, as an indicator of soluble organic materials, in the WAF. A WAF of a nominal loading rate of 100 mg/L was prepared in duplicate in deionised reverse osmosis water and stirred using a stirring rate such that a vortex was formed to give a dimple at the water surface. One loading rate was stirred for a period of 23 hours and the other for a period of 95 hours. After a 1-hour standing period the mixtures were then removed by siphon and samples taken for Total Organic Carbon analysis.
- It was evident from this work that increasing the stirring period did not significantly increase the amount of carbon in the WAF and so preparation of the WAF was maintained at 24 hours.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain/clone: 1st instar Daphnia magna
- Source: in-house laboratory cultures
- Feeding during test: no

ACCLIMATION
- Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water in a temperature controlled room at 18.9 to 19.1 °C. Temperatures at which the stock daphnids were kept were observed to be slightly below the range given in the protocol of 20 ± 1 °C. This deviation was considered not to have affected the outcome or the validity of the test as there was no effect on survival and reproduction of the stock cultures. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
- Type and amount of food: Each culture was fed daily with a suspension of algae (Chlorella sp.).

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
- Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

250 mg/L (CaCO3)

Test temperature:

20.1 - 20.6 °C

pH:

7.8 - 7.9

Dissolved oxygen:

8.6 -9.1 mg O2/L

Nominal and measured concentrations:

Nominal: 100 mg/L WAF

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass jars
- The test vessels were covered to reduce evaporation.
- Fill volume: 200 mL
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Stock Solution A: CaCl2.2H2O 11.76 g/L
Stock Solution B: MgSO4.7H2O 4.93 g/L
Stock Solution C: NaHCO3 2.59 g/L
Stock Solution D: KCl 0.23 g/L
Reconstituted Water: An aliquot (25 mL) of each of solutions A-D was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm^-1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCI and was aerated until the dissolved oxygen concentration was approximately air-saturation value. The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3.
- Intervals of water quality measurement: Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. The pH and dissolved oxygen concentration was measured using a WTW pH/Oxi 3401 pH and dissolved oxygen meter, and the temperature was measured using a Hanna Instruments HI 93510 digital thermometer. The vortex depth was recorded at the start and end of the mixing period.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods

EFFECT PARAMETERS MEASURED
- Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

RANGE-FINDING STUDY
- Test concentrations: In the range-finding test Daphnia magna were exposed to a series of nominal loading rates of 10 and 100 mg/L. The test material was prepared as a WAF.
- An amount of test material (100 and 250 mg) were each separately added to the surface of 10 and 2.5 litres of reconstituted water to give the 10 and 100 mg/L loading rates respectively. After the addition of the test material, the reconstituted water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. These were stirred for 23 hours. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. Microscopic observations made on the W AFs indicated that a significant amount of dispersed test material was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 100 mg/L loading rate WAF. Microscopic observations of the WAF were performed after filtering and showed no micro-dispersions or particles of test material present.
- In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at approximately 20 °C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each 250 mL test and control vessel contained 200 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilised Daphnia magna were recorded.
- Results used to determine the conditions for the definitive study: Yes, based on the results of the range-finding test a "Limit test" was conducted at a single loading rate of 100 mg/L to confirm that no immobilisation or adverse reactions to exposure were observed.

EVALUATION OF DATA
- An estimate of the EL50 values was given by inspection of the immobilisation data.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mobility

Details on results:

Range-finding Test
- No immobilisation was observed at the 10 and 100 mg/L loading rate WAFs.
- Based on this information, a single loading rate of four replicates, of 100 mg/L, using a stirring period of 23 hours followed by a 1-hour standing period, was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that no immobilisation or adverse reactions to exposure were observed.

Definitive Test
Immobilisation data:
- There was no immobilisation in 20 daphnids exposed to a 100 mg/L loading rate WAF for a period of 48 hours. The No Observed Effect Loading rate after 24 and 48 hours exposure was 100 mg/L loading rate WAF. The No Observed Effect Loading rate is based upon zero immobilisation at this loading rate. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L. No immobilisation was observed in the control replicates throughout the test and hence satisfied the validation criterion given in the OECD Guideline which states that immobilisation in the control should not exceed 10 %.
Physico-chemical measurements:
- Temperature was maintained at approximately 20 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.
- The oxygen concentration in one of the test vessels was observed to have an air saturation value (ASV) of 100 %. This was considered to be due to the presence of microscopic air bubbles in the media super-saturating the diluent and was considered not to have had an impact on the outcome or integrity of the test as no adverse effects were observed.
- The dissolved oxygen concentration at the end of the test was 8.6 to 9.1 mg O2/L and hence satisfied the validation criterion given in the OECD Guideline which states that the dissolved oxygen concentration should be a minimum of 3 mg O2/L at the end of the test.
- Vortex depth measurements: The vortex depth was recorded at the start and end of the mixing period and was observed to be a dimple at the water surface on each occasion

Observations on test material solubility:
- Observations on the test media were carried out during the mixing and testing of the WAFs. At the start of the mixing period and after 23 hour stirring the 100 mg/L loading rate was observed to be a clear, colourless water column with brown flakes of test material on the surface and dispersed throughout. After a 1-hour standing period the 100 mg/L loading rate was observed to be a clear, colourless water column with flakes of test material on the surface and bottom of vessel. The WAF was removed by filtering through a glass wool plug (2-4 cm in length). Microscopic examination after filtering showed the glass wool plug had removed all particles of test material. During the test the 100 mg/L loading rate was observed to be a clear, colourless solution.

Chemical analysis of test loading rates:
- Analysis of the test preparations at 0 hours and 48 hours showed measured test concentrations to be less than the limit of quantitation and hence did not provide definitive evidence of stability of the test preparations. Given that toxicity cannot be attributed to a single component or a mixture of components but to the test material as a whole, and the dissolved test material was below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.

Results with reference substance (positive control):

EC50 values:
3 h: > 3.2 mg/L
24 h: 1.2 mg/L (95 % CL: 1.0 - 1.4 mg/L)
48 h: 0.85 mg/L (95 % CL: 0.76 - 0.96 mg/L)

- The No Observed Effect Concentration after 24 and 48 hours was 0.56 mg/L. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.
- The slope and standard error of the response curve at 24 hours was 9 .2 (SE = 2.1 ). Due to the unsuitable nature of the data it was not possible to calculate the slope and standard error of the response curve at 48 hours.
- The results from the positive control with potassium dichromate were within the normal range for this reference material. The mean 48-Hour EC50 value calculated from all positive controls was 0.82 mg/L (sd = 0.22).

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the exposure of the test material to the freshwater invertebrate Daphnia magna gave a 48-Hour EL50 value of greater than 100 mg/L loading rate WAF. Correspondingly the No Observed Effect Loading rate was 100 mg/L loading rate WAF.

Executive summary:

The short-term toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guidelines OECD 202 and EU Method C.2, under GLP conditions.

The study was performed to assess the acute toxicity of the test material to Daphnia magna. Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test material, at a single nominal loading rate of 100 mg/L for 48 hours at a temperature of approximately 20 °C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

A positive control conducted approximately every six months used potassium dichromate as the reference material. Daphnia magna was exposed to an aqueous solution of the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L for 48 hours at a temperature of 20.0 to 21.1 °C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 3, 24 and 48 hours.

The 48-hour EL50 for the test material to Daphnia magna based on nominal loading rates was greater than 100 mg/L loading rate WAF and correspondingly the No Observed Effect Loading rate was 100 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

Analysis of the test preparations at 0 hours and 48 hours showed measured test concentrations to be less than the limit of quantitation and hence did not provide definitive evidence of stability of the test preparations. Given that toxicity cannot be attributed to a single component or a mixture of components but to the test material as a whole, and the dissolved test material was below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.

The 48-hour EC50 for the reference material to Daphnia magna based on nominal concentrations was 0.85 mg/L with 95 % confidence limits of 0. 76 - 0.96 mg/L. The No Observed Effect Concentration was 0.56 mg/L.

Under the conditions of this study, the exposure of the test material to the freshwater invertebrate Daphnia magna gave a 48-hour EL50 value of greater than 100 mg/L loading rate WAF. Correspondingly the No Observed Effect Loading rate was 100 mg/L loading rate WAF.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted on read-across material

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across hypothesis is based on an analogue approach. Within this approach, it is assumed that, based on structural similarity, the source and target substances will display similar toxicological properties. Of the analogue approaches detailed in the RAAF guidance document, Scenario 2 “Assessment of different compounds that have qualitatively similar properties” is considered the most appropriate hypothesis for this read-across. AEs specific to this scenario are considered within the attached read-across justification report.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Both the source and target are long-chain alkenyl succinimides. Both the source and target substances are long-chain alkenyl succinimides. The source substance is suspected to contain lactone acids and ketone acids that are not present in the target substance. However, these substances are not expected to result in a significant difference in aquatic toxicity between the source and target substances.

3. ANALOGUE APPROACH JUSTIFICATION
The source and target substances are highly consistent in their toxicological profiles, with both the source and target demonstrating no toxicity to aquatic organisms.

4. DATA MATRIX
See attached read-across justification report.

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

mobility

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the exposure of the test material to the freshwater invertebrate Daphnia magna gave a 48-Hour EL50 value of greater than 100 mg/L loading rate WAF. Correspondingly the No Observed Effect Loading rate was 100 mg/L loading rate WAF.

Executive summary:

This read-across hypothesis is based on an analogue approach. Within this approach, it is assumed that, based on structural similarity, the source and target substances will display similar toxicological properties. The source and target substances are highly consistent in their toxicological profiles, with both the source and target demonstrating no toxicity to aquatic organisms. Differences between the source and target substances are not anticipated to affect the use of the source substance for read across.

The short-term toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guidelines OECD 202 and EU Method C.2, under GLP conditions. The study was performed to assess the acute toxicity of the test material to Daphnia magna. Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test material, at a single nominal loading rate of 100 mg/L for 48 hours at a temperature of approximately 20 °C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours. A positive control conducted approximately every six months used potassium dichromate as the reference material. Daphnia magna was exposed to an aqueous solution of the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L for 48 hours at a temperature of 20.0 to 21.1 °C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 3, 24 and 48 hours. The 48-hour EL50 for the test material to Daphnia magna based on nominal loading rates was greater than 100 mg/L loading rate WAF and correspondingly the No Observed Effect Loading rate was 100 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L. Analysis of the test preparations at 0 hours and 48 hours showed measured test concentrations to be less than the limit of quantitation and hence did not provide definitive evidence of stability of the test preparations. Given that toxicity cannot be attributed to a single component or a mixture of components but to the test material as a whole, and the dissolved test material was below the quantifiable limit of the analytical method, the results were based on nominal loading rates only. The 48-hour EC50 for the reference material to Daphnia magna based on nominal concentrations was 0.85 mg/L with 95 % confidence limits of 0. 76 - 0.96 mg/L. The No Observed Effect Concentration was 0.56 mg/L. Under the conditions of this study, the exposure of the test material to the freshwater invertebrate Daphnia magna gave a 48-hour EL50 value of greater than 100 mg/L loading rate WAF. Correspondingly the No Observed Effect Loading rate was 100 mg/L loading rate WAF.

Description of key information

Read-across Data

Under the conditions of this study, the exposure of the test material to the freshwater invertebrate Daphnia magna gave a 48-hour EL50 value of greater than 100 mg/L loading rate WAF. Correspondingly the No Observed Effect Loading rate was 100 mg/L loading rate WAF.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

Read-across Data

The short-term toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guidelines OECD 202 and EU Method C.2, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The study was performed to assess the acute toxicity of the test material to Daphnia magna. Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test material, at a single nominal loading rate of 100 mg/L for 48 hours at a temperature of approximately 20 °C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

A positive control conducted approximately every six months used potassium dichromate as the reference material. Daphnia magna was exposed to an aqueous solution of the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L for 48 hours at a temperature of 20.0 to 21.1 °C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 3, 24 and 48 hours.

The 48 -hour EL50 for the test material to Daphnia magna based on nominal loading rates was greater than 100 mg/L loading rate WAF and correspondingly the No Observed Effect Loading rate was 100 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

Analysis of the test preparations at 0 hours and 48 hours showed measured test concentrations to be less than the limit of quantitation and hence did not provide definitive evidence of stability of the test preparations. Given that toxicity cannot be attributed to a single component or a mixture of components but to the test material as a whole, and the dissolved test material was below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.

The 48-hour EC50 for the reference material to Daphnia magna based on nominal concentrations was 0.85 mg/L with 95 % confidence limits of 0. 76 - 0.96 mg/L. The No Observed Effect Concentration was 0.56 mg/L.

Under the conditions of this study, the exposure of the test material to the freshwater invertebrate Daphnia magna gave a 48-hour EL50 value of greater than 100 mg/L loading rate WAF. Correspondingly the No Observed Effect Loading rate was 100 mg/L loading rate WAF.