Amines, N-tallow alkyltrimethylenedi-, C4-18-alkyl phosphates

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

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 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

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 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, the measured concentrations were at the level of nominal loading rates (82-98%), proving correct preparation of the WAFs. In the samples collected from the test solutions, the measured concentrations were 0.0096, 0.027, 0.080, 0.19 and 0.90 mg/L in WAFs prepared at 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L, respectively, at the start of the test. These concentrations were at the level of 98-145% of the initial concentrations 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. Water Accommodated Fractions (WAFs) were individually prepared at loading rates in the range of 0.010 to 1.0 mg/L and used as test concentrations. Test solutions were prepared by using stock solutions in methanol. Since the test item is an UVCB, the solvent was completely evaporated prior to addition of the test medium to avoid modification of WAF-composition due to presence of a water-miscible solvent.
A final test was performed based on the results of a preceding combined limit/range-finding test and range-finding test. Six replicates of exponentially growing algal cultures per group were exposed to a blank and a solvent control. Three replicates per test concentration were exposed to WAFs individually prepared at loading rates of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L. The initial algal cell density was 104 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start and after 72 hours of exposure.

Test organisms

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

Species Raphidocelis subcapitata, strain: NIVA CHL 1
Source In-house laboratory culture.
Reason for selection This system is a unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

Between 22 and 23°C

pH:

6-9

Nominal and measured concentrations:

In the samples collected from the test solutions, the measured concentrations were 0.0096, 0.027, 0.080, 0.19 and 0.90 mg/L in WAFs prepared at 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L, respectively, at the start of the test. These concentrations were at the level of 98-145% of the initial concentrations at the end of the test.

Details on test conditions:

Stock culture Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
Light intensity 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
Stock culture medium M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:
NaNO3 500 mg/L
K2HPO4 39.5 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3 20 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L

Pre-culture 3 (combined limit/range-finding test and range-finding test) and 4 (final test) days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
Pre-culture medium M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition:
NH4Cl 15 mg/L
MgCl2.6H2O 12 mg/L
CaCl2.2H2O 18 mg/L
MgSO4.7H2O 15 mg/L
KH2PO4 1.6 mg/L
FeCl3.6H2O 64 µg/L
Na2EDTA.2H2O 100 µg/L
H3BO3 185 µg/L
MnCl2.4H2O 415 µg/L
ZnCl2 3 µg/L
CoCl2.6H2O 1.5 µg/L
CuCl2.2H2O 0.01 µg/L
Na2MoO4.2H2O 7 µg/L
NaHCO3 50 mg/L
\HEPES buffer 6 mmol/L
Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)
pH 8.1 ± 0.2

Combined Limit/Range-Finding Test
The project started with a combined limit/range-finding test. Six replicates of exponentially growing algae 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:
• Three replicates per concentration 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.
• Cell densities were recorded at 24-hour intervals in the control and the limit concentration. Intermediate concentrations were measured only at the end of the exposure period.
• After 24 hours of exposure, particles were observed at the highest WSF tested that disturbed spectrophotometric measurement of algal cell densities. Therefore the algal cell density was determined by use of a microscope and a counting chamber throughout the study.
• Samples for possible analysis were taken at the start, after 24 hours of exposure and at the end of the test period.
• pH was only measured in the control and the highest test concentration.
• At the end of the test algae were not observed under a microscope to verify a normal and healthy appearance.

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:
• Three replicates of exponentially growing algal cultures 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.
• Cell densities were recorded at 24-hour intervals in the control and the limit concentration. Intermediate concentrations were measured only at the end of the exposure period.
• pH was only measured in the control and the highest test concentration.
• At the end of the test algae were not observed under a microscope to verify a normal and healthy appearance.

Final Test
Test Concentrations
Test item WAFs individually prepared at loading rates of 0.010, 0.032, 0.10, 0.32, and 1.0 mg/L.
Control Test medium after evaporation from the test vessels of the solvent used to prepare the stock solutions (see paragraph 4.5).
Replicates 3 replicates of each test concentration,
6 replicates of the control,
1 replicate of each test concentration without algae.
4.8.3.2. Test Procedure and Conditions
Test duration 72 hours
Test type Static
Test vessels 100 mL, all-glass with aluminium caps, perforated for ventilation, containing 50 mL of test solution
Test Medium M2 (see paragraph 4.7.)
Cell density An initial cell density of 1 x 104 cells/mL.
Illumination Continuously using TLD-lamps with a light intensity within the range of 79 to 82 µE.m-2.s-1.
Incubation Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

0.14 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.032 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.032 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Total inhibition of growth rates and yield was recorded already at the lowest WSF tested.

Based on these results, samples taken from the WSF prepared at loading rates of 1.0 mg/L were analysed by following the di-ester compound of the test item. The concentration measured at the start of the test was 0.44 mg/L. During the exposure period, this concentration remained stable, i.e. was at the level of 102-111% of initially measured through the test.

All test conditions were maintained within the limits prescribed by the study plan.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study with Raphidocelis subcapitata, biologically relevant inhibition of growth rate of this fresh water algae species exposed to Amines, N-tallow alkyltrimethylenedi-, C4-18-alkyl phosphates was observed at loading rates of 0.32 mg/L and higher.
The 72h-EL50 for growth rate inhibition (ERL50) was 0.18 mg/L with a 95% confidence interval ranging from 0.17 to 0.19 mg/L.
The 72h-EL50 for yield inhibition (EYL50) was 0.14 mg/L with a 95% confidence interval ranging from 0.075 to 0.27 mg/L.
The 72h-NOELR for growth rate inhibition was 0.032 mg/L based on statistical significance and 0.10 mg/L based on biological relevance.
The 72h-NOELR for yield inhibition was 0.032 mg/L based on both statistical significance and biological relevance.

GHS classification: 72 or 96 hr ErC50 (for algae or other aquatic plants) = 1 mg/l - Category Acute 1: