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Ecotoxicological information

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:
Updated information. Study now available and provided ahead of extended submission date of 31/05/2019.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Amines, N-tallow alkyltrimethylenedi-, C4-18-alkyl phosphates
EC Number:
271-672-2
EC Name:
Amines, N-tallow alkyltrimethylenedi-, C4-18-alkyl phosphates
Cas Number:
68603-74-7
Molecular formula:
The substance is a UVCB so there is no molecular formula
IUPAC Name:
Amines, N-tallow alkyltrimethylenedi-, C4-18-alkyl phosphates
Test material form:
solid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 30 April 2019
- Stability under test conditions: Yes
- Expiration date of the lot/batch: 30 April 2019
- Solubility and stability of the test substance in the solvent/vehicle: Not applicable.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

Test solutions

Vehicle:
no
Details on test solutions:
The batch of Amines, N-tallow alkyltrimethylenedi-, C4-18-alkyl phosphates tested was an amber greasy solid UVCB which was not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test item.

Preparation of test solutions for the combined limit/range-finding test and range-finding test started with loading rates individually prepared ranging between 1.0 and 100 mg/L. A three day period of magnetic stirring was applied to ensure maximum dissolution of the test item in medium. Thereafter, the aqueous Water Soluble Fractions (WSFs) were collected by filtration through a 0.45 µm membrane filter (RC55, Whatman) and used as test concentrations. As suggested by the OECD series on testing and assessment number 23, 2019, lower test concentrations in the range-finding test were prepared by diluting the WSF prepared at a loading rate of 1.0 mg/L. In fact, WSFs could not be prepared individually due to either the impossibility to weight such small amounts and/or the large test solution volumes eventually required.

Based on the results obtained in the combined limit/range-finding test and range-finding test, the preparation procedure was adjusted for the subsequent final test. Water Accommodated Fractions (WAFs) were prepared by using stock solutions in methanol at nominal concentrations of 2.2, 4.6, 10, 22, 46 and 100 mg/L. Empty test vessels were individually spiked with 0.5 mL of the respective stock. Each replicate of the control received 0.5 mL blank methanol. The solvent was completely evaporated overnight prior to addition of the test medium to avoid modification of WAF-composition due to presence of a water-miscible solvent. Thereafter, 50 mL test medium was added to each vessel. Test solutions, including those of the controls, were agitated for a period of three days to ensure maximum dissolution of test item in test medium.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
Species Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
Source In-house laboratory culture with a known history.
Reason for selection This system has been selected as an internationally accepted invertebrate species.
Validity of batch Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20% , presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
Characteristics Daphnia, less than 24 hours old, from parental daphnids of more than two weeks old.

Study design

Test type:
static
Water media type:
other: Purified tap water
Limit test:
yes
Total exposure duration:
48 h

Test conditions

Hardness:
CaCO3: 180 mg/L
Test temperature:
Between 19 and 20°C
pH:
Between 6 and 9.
Dissolved oxygen:
The dissolved oxygen concentration at the end of the test was =3 mg/L in control and test vessels.
Nominal and measured concentrations:
The measured concentrations were 0.016, 0.044, 0.071, 0.17, 0.25 and 0.65 mg/L in Water Accommodated Fractions prepared at 0.022, 0.046, 0.10, 0.22, 0.46 and 1.0 mg/L, respectively, at the start of the test. These concentrations were at the level of 66-131% of the initial concentrations at the end of the test.
Details on test conditions:
Breeding
Start of each batch Approximately 250 newborn daphnids, i.e. less than 3 days old, were placed into 5 litres of medium in an all-glass culture vessel.
Maximum age of the cultures 4 weeks
Renewal of the cultures After 7 days of cultivation, half of the medium twice a week.
Temperature of medium 18-22°C
Feeding Daily, a suspension of fresh water algae.
Culture medium M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).

The following salts and vitamins were added to freshly prepared test medium (see section 4.8.3.2) to reach the following concentrations:

Salts: H3BO3 0.71 mg/L
FeSO4.7H2O 0.25 mg/L
MnCl2.4H2O 0.090 mg/L
LiCl 0.076 mg/L
RbCl 0.018 mg/L
SrCl2.6H2O 0.038 mg/L
Na2MoO4.2H2O 0.015 mg/L
NaBr 0.0040 mg/L
CuCl2.2H2O 0.0042 mg/L
ZnCl2 0.013 mg/L
CoCl2.6H2O 0.010 mg/L
KI 0.0032 mg/L
Na2SeO3 0.0022 mg/L
NH4VO3 0.00057 mg/L
Na2EDTA.2H2O 0.62 mg/L
Na2SiO3.5H2O 7.5 mg/L
NaNO3 0.27 mg/L
KH2PO4 0.14 mg/L
K2HPO4 0.18 mg/L
Vitamins: Thiamine hydrochloride 75.0 µg/L
B12 1.0 µg/L
Biotin 0.75 µg/L

Combined Limit/Range-Finding Test
The project started with a combined limit/range-finding test. Twenty daphnids per concentration (four replicates, 5 daphnids per vessel) were exposed to an untreated control and a WSF prepared at a loading rate of 100 mg/L. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
• Ten daphnids per concentration (in duplicate, 5 per vessel) were exposed to WSFs individually prepared at loading rates of 1.0 and 10 mg/L in the combined range-finding test. No solvent was used to facilitate dissolution of the test item in test medium.
• Dissolved oxygen concentrations and pH were only measured in the control and the highest test concentration.

Range-Finding Test
Based on the results of the combined limit/range-finding test, an additional range-finding test was performed to provide information about the range of concentrations to be tested in the final test. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
• Ten daphnids per concentration (in duplicate, 5 per vessel) were exposed to an untreated control and to test solutions containing 0.10, 1.0, 10 and 100% of a WSF prepared at a loading rate of 1.0 mg/L. No solvent was used to facilitate dissolution of the test item in test medium.
• Dissolved oxygen concentrations and pH were only measured in the control and the highest test concentration.
Reference substance (positive control):
no

Results and discussion

Effect concentrations
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
0.27 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
The results of analysis of the samples taken during the final test are described in Table 4 of the appended Analytical Report.

Samples taken from all stock solutions, test concentrations and the control were analysed by following the di-ester compound of the test item. In the samples collected from the stock solutions at the start of the test, the measured concentrations were at the level of 79-95% of the nominal loading rates, proving correct preparation of the WAFs. At the start of the test, the measured concentrations were 0.016, 0.044, 0.071, 0.17, 0.25 and 0.65 mg/L in WAFs prepared at 0.022, 0.046, 0.10, 0.22, 0.46 and 1.0 mg/L, respectively. These concentrations were at the level of 66-131% of the initial concentrations at the end of the test. At the end of the test, the concentration analysed in the extra sample collected from the WAF prepared at a loading rate of 0.22 mg/L was 0.28 mg/L, while the concentration analysed in the remaining three pooled replicates was 0.17 mg/L.

It should be noted that a response was recorded in the control at the start and at the end of the test. The reason is unknown. However, since no immobility or other signs of disease or stress was observed in the control throughout the test, it can be it can be stated that this has no influence on the results of the test.

Since the test item is a UVCB and measurements were based only on the di-ester compound in the test item, it is unknown whether the analytically monitored constituent is responsible for the recorded effects. Therefore, effect parameters were expressed based on loading rates.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The 48h-EL50 for Daphnia magna exposed to Amines, N-tallow alkyltrimethylenedi-, C4-18-alkyl phosphates was 0.27 mg/L (95% confidence interval between 0.22 and 0.32 mg/L) and was already reached after 24 hours of exposure.