1-(bis(2-(1,3-dimethylbutylideneamino)ethyl)amino)-3-phenoxypropan-2-ol

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:

05-Jul-2018 to 01-Feb-2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1-(bis(2-(1,3-dimethylbutylideneamino)ethyl)amino)-3-phenoxypropan-2-ol

EC Number:

271-340-7

EC Name:

1-(bis(2-(1,3-dimethylbutylideneamino)ethyl)amino)-3-phenoxypropan-2-ol

Cas Number:

68541-07-1

Molecular formula:

C25H43N3O2

IUPAC Name:

1-(bis(2-(1,3-dimethylbutylideneamino)ethyl)amino)-3-phenoxypropan-2-ol

Test material form:

liquid

Specific details on test material used for the study:

Appearance: Light yellow liquid
Batch: UL18401660
Purity/Composition: ca. 91.48%
Test item storage: At room temperature
Stable under storage conditions until: 15 April 2020 (expiry date)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.

Frequency at t=0 h and t=48 h Volume 2.4 mL from the approximate centre of the test vessels Storage Not applicable, samples were transferred to the analytical laboratory at the Test Facility and analysed on the day of sampling. At the end of the exposure period, the replicates were not pooled at each concentration before sampling. Instead, samples were taken from one test vessel per concentration.

Test solutions

Vehicle:

no

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Test System 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%1, 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.

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 allglass 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).

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

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

The hardness of test medium expressed as CaCO3: 180 mg/L

Test temperature:

between 19 and 20°C

pH:

pH between 6 and 9

Dissolved oxygen:

oxygen: >= 3 mg/L at the end of the test

Nominal and measured concentrations:

Measured
The actual test item concentrations based on the measurements of the ketone component were 9.5, 17, 31, 53 and 95 mg/L at the WAFs prepared at 10, 18, 32, 56 and 100 mg/L respectively

The actual test item concentrations based on the measurements of the diamine component were 7.9, 15, 27, 42 and 83 mg/L at the WAFs prepared at 10, 18, 32, 56 and 100 mg/L respectively

the 48h-EC50 for Daphnia magna exposed to 1-(bis(2-(1,3dimethylbutylideneamino) ethyl)amino)-3-phenoxypropan-2-ol was 17 mg/L based on measured concentrations (95% confidence interval between 14 and 20 mg/L).

Nominal
QC Samples at 219 nm

Test item conc. for purity (mg test item/L)

Target Nominal
0 0
5 5
100 100

Details on test conditions:

TEST SYSTEM

Test duration 48 hours Test type Static Test vessels 60 mL, all-glass airtight closed without headspace. Test medium The following salts (analytical grade) were added to tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands): CaCl2.2H2O 211.5 mg/L MgSO4.7H2O 88.8 mg/L NaHCO3 46.7 mg/L KCl 4.2 mg/L The hardness of test medium expressed as CaCO3: 180 mg/L with a pH between 6 and 9. Number of daphnids 20 per concentration Loading 5 per vessel containing 60 mL of test solution. Light A daily photoperiod of 16 hours. Feeding No feeding Aeration No aeration of the test solutions was applied. Introduction of daphnids Within 20 minutes after preparation of the test solutions.

Test medium The following salts (analytical grade) were added to tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands): CaCl2.2H2O 211.5 mg/L, MgSO4.7H2O 88.8 mg/L, NaHCO3 46.7 mg/L, KCl 4.2 mg/L. The hardness of test medium expressed as CaCO3: 180 mg/L with a pH between 6 and 9.

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 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

Light: A daily photoperiod of 16 hours

EFFECT PARAMETERS MEASURED:
Immobility (including mortality) At 24 hours and at 48 hours.
pH and dissolved oxygen At the beginning and at the end of the test, for all concentrations and the control.
Temperature of medium Continuously in a temperature control vessel, beginning at the start of the test.

Control: Test medium without test item or other additives

RANGE-FINDING STUDY
- Test concentrations: Ten daphnids per concentration (in duplicate, 5 per vessel) were exposed to loading rates individually prepared at 1.0, 10 and 100 mg/L and to a control.
- A final test was performed based on the results of a range-finding test. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to WAFs individually prepared at loading rates of 10, 18, 32, 56 and 100 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (K2Cr2O7, art. 1.04864, batch no. K50664264)

Results and discussion

Effect concentrations

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

ca. 17 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Range-Finding Test
No immobility was observed in the control and the two lowest test concentrations throughout the test. At the end of the test, complete immobility was observed at the highest test concentration. Therefore, the expected EC50 was between loading rates of 10 and 100 mg/L. All test conditions were maintained within the limits prescribed by the study plan.

Measured Concentrations
Samples taken from all test concentrations and the control were analysed. Separate analysis of the hydrolysed components of the ketone (219 nm) and the diamine (243 nm) were performed for each sample. The actual test item concentrations based on the measurements of the ketone component were 9.5, 17, 31, 53 and 95 mg/L at the WAFs prepared at 10, 18, 32, 56 and 100 mg/L respectively, at the start of the test. During the exposure period, the concentrations remained stable, i.e. were at 98-100% relative to initial at the end of the test. The actual test item concentrations based on the measurements of the diamine component were 7.9, 15, 27, 42 and 83 mg/L at the WAFs prepared at 10, 18, 32, 56 and 100 mg/L respectively, at the start of the test. During the exposure period, the concentrations remained stable, i.e. were at 94-95% relative to initial at the end of the test. It should be noted that a response was measured in the control. However, since this response was not measured based on the ketone component, and approximately the same response was measured in the QC blanc sample it was considered the response was not from the hydrolysis product of the test item and therefore has no impact on the study results.

Based on these results, effect parameters were expressed as initially measured concentrations of the diamine component. The analytical results of the diamine component were used since they are lower than the analysed concentrations of the ketone component and thus are seen as the worst case scenario.

Immobility
No immobility was observed in the control and the lowest test concentration throughout the exposure period. At the end of the test, a dose-related increase of immobility was observed at measured concentrations of 15 mg/L and higher, reaching 100% immobility at the two highest test concentrations.

Experimental Conditions
These test conditions remained within the limits prescribed by the study plan (pH: 6-9, not varying by more than 1.5 units; oxygen: 3 mg/L at the end of the test). It should be noted that the pH of the WAF prepared at a loading rate of 56 mg/L was adjusted from 9.1 to 8.5 at the start of the test using 1 mol HCl/L (Merck, Darmstadt, Germany). The temperature continuously measured in a temperature control vessel varied between 19 and 20°C during the test, and complied with the requirements as laid down in the study plan (18-22°C, constant within ±1°C).

Results with reference substance (positive control):

The actual responses in this reference test with K2Cr2O7 are generally within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was between 0.28 and 0.90 mg/L. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at Charles River Den Bosch.

Reported statistics and error estimates:

The 24 and 48h-EC50-values were calculated from the probits of the percentages of affected daphnids and the logarithms of the corresponding test item concentrations (initially measured) using the maximum likelihood estimation method. ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, the 48h-EC50 for Daphnia magna exposed to 1-(bis(2-(1,3dimethylbutylideneamino) ethyl)amino)-3-phenoxypropan-2-ol was 17 mg/L based on measured concentrations (95% confidence interval between 14 and 20 mg/L).

Executive summary:

The objective of the study was to evaluate 1-(bis(2-(1,3-dimethylbutylideneamino)ethyl)amino)-3-phenoxypropan-2-ol for its ability to generate acute toxic effects on the

mobility of Daphnia magna during an exposure period of 48 hours and, if possible, to determine the EC50 at 24 and 48 hours of exposure. The study procedures described in this report were based on the OECD guideline No. 202, 2004. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, July 6, 2018. The batch of 1-(bis(2-(1,3-dimethylbutylideneamino) ethyl)amino)-3-phenoxypropan-2-ol tested was a light yellow liquid with a purity of 91.48% and not completely soluble in test medium at the loading rates initially prepared. A correction was made for the purity/composition of the test item. A correction factor of 1.09 was used. All further concentrations reported are based on pure test item. All glassware used during the preparation of test solutions was closed with minimal headspace in order to minimize vaporization of the test item.

A final test was performed based on the results of a range-finding test. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to WAFs individually prepared at loading rates of 10, 18, 32, 56 and 100 mg/L. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test. Samples taken from all test concentrations and the control were analysed. Separate analysis of the hydrolysed components of the ketone (219 nm) and the diamine (243 nm) were performed for each sample. The actual test item concentrations based on the measurements of the ketone

component were 9.5, 17, 31, 53 and 95 mg/L at the WAFs prepared at 10, 18, 32, 56 and 100 mg/L respectively, at the start of the test. During the exposure period, the concentrations remained stable, i.e. were at 98-100% relative to initial at the end of the test. The actual test item concentrations based on the measurements of the diamine component were 7.9, 15, 27, 42 and 83 mg/L respectively, at the start of the test. During the exposure period, the concentrations remained stable, i.e. were at 94-95% relative to initial at the end of the test.

Based on these results, effect parameters were expressed as initially measured concentrations of the diamine component. No immobility was observed in the control and the lowest test concentration throughout the exposure period. At the end of the test, a dose-related increase of immobility was observed at measured concentrations of 15 mg/L and higher, reaching 100% immobility at the two highest test concentrations. The study met the acceptability criteria prescribed by the study plan and was considered valid.

In conclusion, the 48h-EC50 for Daphnia magna exposed to 1-(bis(2-(1,3dimethylbutylideneamino) ethyl)amino)-3-phenoxypropan-2-ol was 17 mg/L based on measured concentrations (95% confidence interval between 14 and 20 mg/L).