Calcium bis[(Z)-N-methyl-N-(1-oxo-9-octadecenyl)aminoacetate]

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

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: SOAP 005-2
- Expiration date of the lot/batch: 15 October 2022
- Purity test date: Not reported


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (nominal 5-30 °C) under nitrogen in a desiccator.
- Stability under test conditions: Stable

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples for analysis were taken from all test concentrations.
- Sampling method: Samples for analysis of test item concentration were taken from the remaining bulk freshly prepared test item solutions in each treatment group at the start of the test. The samples for analysis of concentration at the end of the test were taken from a pooled samples of the four treatment group replicates for each exposure concentration.
- Sample storage conditions before analysis: All samples were stored frozen until required for analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A concentrated stock solution was prepared by diluting 50.45 mg of the test item in 500 mL of Elendt M4 medium. The stock solution was ultrasonicated for twenty minutes, and then diluted with Elendt M4 medium to prepare a the highest nominal concentration of 10 mg/L. The remaining test solutions were prepared by serial dilution with Elendt M4 medium (geometric factor: 2.2).
- Controls: Test medium without test item was used as a control.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The concentrated stock solution was observed to be slightly opaque but appeared homogeneous. The test solutions were clear and colourless with no undissolved particulates.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia
- Age at test initiation: less than 24 hours old and not first brood progeny
- Culture conditions: Similar to test
TEST ORGANISM
- Common name: Waterflea
- Age at study initiation (mean and range, SD): Less than 24 hours old and not of first brood progeny.
- Source: Daphnids were originally obtained from Sciento Ltd, UK and maintained at the testing facility.
- Feeding during test : No

ACCLIMATION
- Acclimation period: Stock cultures of Daphnia were kept at the same incubation conditions as required for the test.
- Acclimation conditions (same as test or not): The same as required for the test.
- Type and amount of food: Daphnids were fed with an aqueous suspension of the unicellular green algae Chlorella vulgaris.
- Feeding frequency: Once daily.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

246 mg/L as CaCO3

Test temperature:

18.1 – 18.7°C (mean: 18.4°C)

pH:

7.65 to 7.72

Dissolved oxygen:

Test start: 9.19 - 9.34 mg/L
Test end: 8.72 - 8.81

Salinity:

N/A

Nominal and measured concentrations:

Nominal concentrations: 0, 0.43, 0.94, 2.1, 4.5, 10 mg/L
Geometric mean measured concentrations: 0.097, 0.15, 0.81, 5.0 and 3.3 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers
- Type: Test vessels were covered with loosely-fitting lids in order to reduce evaporation and prevent dust entering the solutions
- Material, size, headspace, fill volume: 250 mL glass vessel, filled with 100 mL of solution
- Aeration: No aeration
- 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: Elendt M4
- Culture medium different from test medium: No
- Intervals of water quality measurement: The temperature, pH and dissolved oxygen were determined in each exposure concentration at the start of the test and from the pooled replicates at the end of the test period. The temperature of the test area was monitored continuously using a data logger. Hardness (as CaCO3) was determined in the bulk dilution medium before use.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light / 8 hours dark, cycle
- Light intensity: 649 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Assessments were made after 24 and 48 hours (± 1 hour). The number of immobilised daphnids at each time point was recorded.


RANGE-FINDING STUDY
- Test concentrations: 0.1, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: 0% immobilisation observed at 0, 0.1 and 1.0 mg/L, 100 % immobilisation at 10 and 100 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (separate test, conducted in August 2017)

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.95 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL; 2.68 - 3.26 mg/L

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.21 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 1.62 - 2.89 mg/L

Details on results:

- Behavioural abnormalities: No incidences of stress of unusual behaviour were observed in the control at 48 hours.
- Mortality of control: No mortalities in the control were observed at 48 hours.
- Immobilisation of control: No immobilisation in the control was observed at 48 hours.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: The 48 hr EC50 (immobilisation) was 1.422 mg/L, within the range of 0.6 – 2.1 mg/L specified in the testing guideline.

Reported statistics and error estimates:

Statistical analysis was carried out using CETIS v 1.8.7.14 software. To calculate the 24 and 48 hours ECx values for immobilisation of Daphnia based on nominal concentrations a 3 Parameter Cumulative log-normal (Probit) test was used. The 24 hours ECx values based on measured concentrations were calculated using a 2 Parameter Log-Gompertz EV test and the 48 hours ECx values were determined using a 2 Parameter Single First Order test.

Data for the highest treatment group of 3.3 mg/L (nominally 10 mg/L) were not included in the calculation of the 48-h EC50 based on geometric mean measured concentrations, as the dose-response inversion in the mean measured concentrations made it impossible to fit a model adequately. The analytical recovery in the highest test concentration (nominal 10 mg/L) sample was 33.3 % of nominal test concentrations, while it was 110.7 % in the next lower test concentration (nominal of 4.5 mg/L). This was considered to be due to the difficulties with analysis of the test item. Additional investigations conducted by the analytical laboratory did not indicate that the analytical recoveries could be improved. The highest treatment group showed a 100% immobilisation while the next lower test concentration exhibited 90% immobilisation, which was in line with the expected response based on the nominal test concentrations. 

Table 1. The results of the definitive Daphnia test

Nominal concentration (mg/L)	24-hour immobilisation (%)	48-hour immobilisation (%)
0 (control)	0	0
0.43	0	0
0.94	0	0
2.1	0	15
4.5	50	90
10	55	100

Table 2: Calcium bis[(Z)-N-methyl-N-(1-oxo-9-octadecenyl) aminoacetate] analysis

Nominal test item concentrations Analytical recoveries expressed as test item mg/L Geometric mean (mg/L) Geometric mean as % nominal T0 hours T48 hours  mg/L mg/L % nominal mg/L % nominal Control 0.02^a N/A 0.04^a N/A N/A N/A 0.43 0.19 44.2 0.03^b NC 0.097 22.7 0.94 0.45 47.9 0.06^b NC 0.15 16.0 2.1 1.99 94.8 0.33 15.7 0.81 38.6 4.5 7.11 158 3.49 77.6 5.0 110.7 10 4.55 45.5 2.44 24.4 3.3 33.3

N/A Not applicable. The limit of quantification (LOQ) for calcium bis[(Z)-N-methyl-N-(1-oxo-9-octadecenyl) aminoacetate] was 0.1 mg/L and the Limit of Detection (LOD) was 0.002 mg/L

NC Not calculable

aCalcium bis[(Z)-N-methyl-N-(1-oxo-9-octadecenyl) aminoacetate] was detected in the controls, however this was < LOQ.

bThese results were below the limit of quantification of the analytical method. These have been substituted with values equivalent to 50% of the LOQ for calculations of the geometric mean measured concentrations.

Validity criteria fulfilled:

yes

Remarks:

In the control, 0% of the daphnids were immobilised at 48 hours; the dissolved oxygen at the end of the test was 8.72 - 8.81 mg/L.

Conclusions:

Based on nominal concentrations, the 48-hour EC50 for Daphnia magna, was 2.95 mg/L. Based on geometric mean measured concentrations, the 48-hour EC50 was 2.21 mg/L.

Executive summary:

A study was performed according to the OECD guideline 202 to assess the effects of the test item, calcium bis[(Z)-N-methyl-N-(1-oxo-9-octadecenyl)aminoacetate], on the immobilisation of Daphnia magna (Cockroft 2019).

Daphnids were exposed to five nominal concentrations of calcium bis[(Z)-N-methyl-N-(1-oxo-9-octadecenyl) aminoacetate], 0.43, 0.94, 2.1, 4.5, 10 mg/L and a control, for 48 hours under static conditions. The test item concentrations were analysed using a LC-MS MRM method and the geometric mean measured concentrations were 0.097, 0.15, 0.81, 5.0 and 3.3 mg/L. As analytical recoveries were outside the range of 100% ± 20% of nominal, study endpoints were expressed in terms of both nominal concentration and geometric mean measured concentrations. The data were statistically analysed with CETIS v1.9.2.7 statistical software package.

All validity criteria for the definitive test were met. Based on nominal concentrations, the 48-hour EC50 to Daphnia magna, was 2.95 mg/L (95% CL: 2.68-3.26 mg/L). Based on geometric mean measured concentrations, the 48-hour EC50 was 2.21 mg/L (95% CL: 1.62-2.89 mg/L).

The study was conducted according to the OECD guideline and is GLP-compliant, therefore is reliable without restrictions and is considered as suitable as a key study for this endpoint.

Description of key information

Based on nominal concentrations, the 48-hour EC50 for immobilisation of Daphnia magna was 2.95 mg/L. Based on geometric mean measured concentrations, the 48-hour EC50 was 2.21 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

2.21 mg/L

Additional information

A study was performed according to the OECD guideline 202 to assess the effects of the test item on immobilisation of Daphnia magna (Cockroft 2019). Five daphnids per test vessel were exposed for 48 hours under static conditions. Based on range-finder results, five nominal concentrations were tested, 0.43, 0.94, 2.1, 4.5 and 10 mg/L (corresponding to geometric mean measured concentrations of: 0.097, 0.15, 0.81, 5.0 and 3.3 mg/L). All validity criteria for the definitive test were met. Analytical recoveries were outside the range of 100% ± 20% of nominal, therefore study endpoints were expressed in terms of both nominal concentration and geometric mean measured concentrations.

The 48-hour EC50 for immobilisation of Daphnia magna was 2.95 mg/L based on nominal concentrations, and based on geometric mean measured concentrations, was 2.21 mg/L. The study was conducted according to the OECD guideline and is GLP-compliant, therefore it is reliable without restrictions and is considered as suitable as a key study for this endpoint.