Amides, tall-oil fatty, N,N'-(iminodi-2,1-ethanediyl)bis-, phosphates

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:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

April 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Duplicate samples were taken from each test concentration and the control at the start and at the end of the two 24-hour test medium renewal periods. For the 24-hour stability samples, the contents of the respective replicates were combined prior to sampling.
All samples were stored deep-frozen (at about -20 °C) immediately after sampling.

Vehicle:

no

Details on test solutions:

- Method: water accommodated fractions (WAFs)
Supersaturated solutions were prepared by dispersing/dissolving the test item into the test water (ISO 6341) at nominal concentrations of 100, 32, 10 and 3.2 mg/L. Ultrasonic treatment for 15 minutes was applied and thereafter, the dispersions were stirred for 48 hours at room temperature in the dark. The stirring period of 48 hours was considered reasonable to achieve maximum solubility for the different components of the test item.
Then, the dispersions were filtered through membrane filters (Whatman, Type NC45, pore size 0.45 µm). The filters were pre-conditioned with 300 mL filtrate. The undiluted filtrates were tested as WAF. The lowest nominal test concentration of 1.0 mg/L was prepared by diluting the 3.2 mg/L WAF.
The method of preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.
- Controls: test water (ISO 6341)

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flee
- Strain/clone: STRAUSS
- Source: in-house culture
- Feeding during test: none

ACCLIMATION
- Acclimation period: none. Breeding was performed in reconstituted water (Elendt M7 Medium), temperature and light conditions were identical to those in the test.
- Type and amount of food: green algae suspension (Desmodesmus subspicatus) or a mixture of this suspension and a commercial fish diet (TETRA MIN Hauptfutter, TETRA-Werke, 49304 Melle / Germany), three times a week
- Health during acclimation (any mortality observed): no mortality or signs of stress were observed and the brood stock was healthy.

The daphnids used for the test were 6 - 24 hours old and were not first brood progeny.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

2.5 mM (250 mg CaCO3/L)

Test temperature:

21 °C

pH:

7.8 - 8.0

Dissolved oxygen:

≥ 8.0 mg O2/L

Nominal and measured concentrations:

nominal loading rates: 1.0, 3.2, 10, 32, and 100 mg/L as WAF
measured test concentrations: below the limit of quantification (LOQ) of 1 mg/L.
The concentrations of the test item were analytically measured in one of the duplicate test media samples from the loading rates of 32 and 100 mg/L and the control from the start and end of both test medium renewal periods. The samples of the lower loading rates (WAF 1.0 to 10 mg/L) were not analyzed since these loading rates were below the 48-hour NOELR determined in this test and were therefore not relevant for the interpretation of the biological results.
The biological results are based on the loading rate of the test item.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL glass beakers containing 50 mL test medium, covered with disposable polypropylene sheets
- Aeration: none
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: 10 mL/animal

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water (ISO 6341) according to the guidelines. Analytical grade salts and additives were dissolved in ultrapure water.
- Ca/Mg ratio: 4:1
- Alkalinity: 0.8 mM
- Culture medium different from test medium: yes (daphnids were cultured in Elendt M7 Medium)
- Intervals of water quality measurement: water temperature, oxygen concentration and pH were measured at the start and at the end each test medium renewal period. At the same dates the appearance of the test media was recorded.

OTHER TEST CONDITIONS
- Adjustment of pH: not necessary
- Photoperiod: 16-hour light to 8-hour dark cycle with a 30-minute transition period
- Lighting: between approximately 15 and 17 µE m-2 s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Daphnids were inspected for immobility and sub-lethal effects (discolouration, surface trapping, incoherent swimming pattern, etc.) after 24 and 48 hours of exposure.

RANGE-FINDING STUDY
- Test concentrations: nominal loading rates (WAF) of 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: no toxic effects up to and including nominal 100 mg/L after 48 hours. However, 100% mortality was observed in a limit test based on the range-finder results and a full test was performed.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

57 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nominal loading rates

Basis for effect:

mobility

Remarks on result:

other:

Remarks:

95% confidence interval: 32 - 100 mg/L

Key result

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nominal loading rates

Basis for effect:

other:

Remarks:

mobility and sub-lethal effects

Details on results:

Behavioural abnormalities:
- Other biological observations: see attached document "Table Effects on Daphnids.pdf"
- Mortality of control: none
- Other adverse effects control: none
- Abnormal responses: none
- Effect concentrations exceeding solubility of substance in test medium: Yes, the solubility of the test item in test water was below the LOQ of the analytical method (< 1 mg/L).

All test media were clear solutions throughout the test period.

For details see attached document "Table Summary of Effect Values on Daphnids.pdf"

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels:
- 24-hour EC50: 1.6 mg/L (within the range 0.6 – 2.1 mg/L as given by the guidelines)

Reported statistics and error estimates:

The 24-hour EL50 of the test item could not be calculated, because none of the responses exceeded 50%. The EL50 was, therefore, also determined directly from the raw data. The 48-hour EL50 could not be calculated by Probit Analysis or Moving Average Interpolation due to the steep concentration-effect relationship. Instead, the 48-hour EL50 was determined as the geometric mean value of the two consecutive loading rates with 0 and 100% immobility (EL0 and EL100), and with confidence limits for the EC50 corresponding to the EL0 and EL100 values. The NOELR, EL0, LOELR and EL100 were determined directly from the raw data.

Validity criteria fulfilled:

yes

Remarks:

In the control, not more that 10 % of the daphnids have been immobilised or showed other sub-lethal effects; the dissolved oxygen concentration at the end of the test was > 3 mg/l in all vessels.

Description of key information

effect value for Daphnia magna, based on loading rates (OECD 202, EU C.2):

48-h EL50: 57 mg/L (solubility in test water < 1 mg/L)

Additional information

The results indicate that the test material is not toxic to daphnia up to the limits of its solubility (< 1 mg/L in test water) and a NOEC and thus a PNEC cannot be determined.