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Diss Factsheets

Ecotoxicological information

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:
04 Jul 2018 to 30 Nov 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
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: 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, t=24 h and t=72 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 with algae were not pooled at each concentration before sampling. Instead, samples were taken from one test vessel per concentration.

Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at a WAF prepared at a loading rate of 1.0 mg/L but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Test solutions

Vehicle:
no
Details on test solutions:
Preparation of Test Solutions 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. Preparation of test solutions started with loading rates individually prepared at a range of 0.22 – 100 mg/L. The test item was weighed on watch glasses which were added to the medium. An overnight period of magnetic stirring was applied to ensure maximum hydrolysis of the test item in its two separate components and maximum dissolution of the components in medium. The obtained mixtures were allowed to settle for a period of one hour. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning and used as test concentrations. All test solutions were clear and colorless at the end of the preparation procedure. After preparation, volumes of 40 mL were added to each replicate of the respective test concentration. Subsequently, 0.8 mL of an algal suspension was added to each replicate providing a cell density of 10 to the power of 4 cells/mL. Any residual volumes were discarded.

Test organisms

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
Species: Raphidocelis subcapitata (formerly known as Pseudokirchneriella 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.

3 - 4 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 10 to the power of 4 cells/mL. The pre-culture was maintained under the same conditions as used in the test.

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:
During the exposure period the temperature measured in the incubator was maintained between 22 and 23°C. Temperature remained within the limits prescribed by the study plan (21-24°C, constant within ±1°C).
pH:
The pH was within the limits prescribed by the study plan (6-9
Nominal and measured concentrations:
Measured Test Item Concentrations:The actual test item concentrations based on the measurements of the ketone component were 0.57, 1.1, 1.2, 2.4, 4.6 and 9.5 mg/L at the WAFs prepared at 0.22, 0.46, 1.0, 2.2, 4.6 and 10 mg/L respectively, at the start of the test. At the end of the test, the two lowest test item concentrations were not detected. The 1.0 mg/L WAF decreased to 54% of initial at the end of the test, while higher concentrations remained stable (i.e. were at 85 – 100% of initial). The actual test item concentrations based on the measurements of the diamine component were 0.47, 0.61, 0.82, 1.9, 3.8 and 8.1 mg/L at the WAFs prepared at 0.22, 0.46, 1.0, 2.2, 4.6 and 10 mg/L,at the start of the test. During the exposure period, the concentrations decreased to 24-92% of initial at the end of the test. 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 blank sample it was considered the response was not from the hydrolysis product of the test item. Based on these results, the Time Weighted Average (TWA) concentrations were calculated. The diamine component was used to express effect parameters. The analytical results of the diamine component were used as they are lower than the analysed concentrations of the ketone component. However, for calculating the ECx-values the two lowest test item concentrations were removed from calculations. These analysed test item concentrations were assumed to not be test item related because they were comparable to the QC blank response. The concentrations measured in the samples taken from solutions with algae were comparable with the concentrations measured in the samples without algae except for the ketone at the end of the test (i.e. t=72 h). Hence, it can be stated that the presence of the algae did affect the concentration of the ketone part but not the diamine part in test medium throughout 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 (tapwater 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 - 4 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 10 to the power of 4 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: Adjusted 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 300 mg/L
HEPES buffer 6 mmol/L
Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)
pH 7.1 ±0.3

Range-Finding Test
A range-finding test was performed to provide information about the range of concentrations to be used 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 loading rates individually prepared at 1.0, 10 and 100 mg/L and to a control. Cell densities were recorded 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. No sampling for determination of actual test concentrations was performed.

Test Concentrations
Test item WAFs individually prepared at loading rates of 0.22, 0.46, 1.0, 2.2, 4.6 and 10 mg/L. Control(s) Test medium without test item or other additives. Replicates 3 replicates of each test concentration, 6 replicates of the control, 1 extra replicate of each test group for sampling purposes after 24 hours of exposure, 1 or 2 replicates of each test concentration without algae.

Test Procedure and Conditions
Test duration: 72 hours
Test type: Static Test vessels 40 mL, airtight closed with no headspace to prevent any loss of the test item due to volatilization.
Test Medium: Adjusted M2.
Cell density: An initial cell density of 1 x 10 to the power of 4 cells/mL.
Illumination: Continuously using TLD-lamps with a light intensity within the range of 82 to 87 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):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 2.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
with a 95% confidence interval ranging from 2.3 to 2.5 mg/L.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 1.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
with a 95% confidence interval ranging from 1.1 to 1.2 mg/L.
Basis for effect:
other: Yield inhibition
Key result
Duration:
72 h
Dose descriptor:
NOEC
Remarks:
based on statistical significance and 0.90 mg/L based on biological relevance.
Effect conc.:
< 0.35 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Growth rate inhibition
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 0.35 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
based on statistical significance and biological relevance.
Basis for effect:
other: yield inhibition
Details on results:
Range-Finding Test
Mean Cell Densities, Inhibition of Growth Rate and Inhibition of Yield
Dose-related inhibition of algal growth rate and yield was found at all test concentrations at the end of the test. An inhibition of 87 and 99% was observed at the highest test concentration, respectively. All test conditions were maintained within the limits prescribed by the study plan.

Final Test
Measured Test Item 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 0.57, 1.1, 1.2, 2.4, 4.6 and 9.5 mg/L at the WAFs prepared at 0.22, 0.46, 1.0, 2.2, 4.6 and 10 mg/L respectively, at the start of the test. At the end of the test, the two lowest test item concentrations were not detected. The 1.0 mg/L WAF decreased to 54% of initial at the end of the test, while higher concentrations remained stable (i.e. were at 85 – 100% of initial). The actual test item concentrations based on the measurements of the diamine component were 0.47, 0.61, 0.82, 1.9, 3.8 and 8.1 mg/L at the WAFs prepared at 0.22, 0.46, 1.0, 2.2, 4.6 and 10 mg/L respectively, at the start of the test. During the exposure period, the concentrations decreased to 24 - 92% of 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 blank sample it was considered the response was not from the hydrolysis product of the test item. Based on these results, the Time Weighted Average (TWA) concentrations were calculated. The diamine component was used to express effect parameters. The analytical results of the diamine component were used since they are lower than the analysed concentrations of the ketone component. However, for calculating the ECx-values the two lowest test item concentrations were removed from calculations. These analysed test item concentrations were assumed to not be test item related because they were comparable to the QC blank response. The concentrations measured in the samples taken from solutions with algae were comparable with the concentrations measured in the samples without algae except for the ketone at the end of the test (i.e. t=72 h). Hence, it can be stated that the presence of the algae did affect the concentration of the ketone part but not the diamine part in test medium throughout the test.

Inhibition of Growth Rate and Inhibition of Yield
Inhibition of growth rate and yield increased with increasing concentrations of 1-(bis(2-(1,3dimethylbutylideneamino) ethyl)amino)-3-phenoxypropan-2-ol at all concentrations tested resulting in 100% inhibition at the highest concentration tested. Statistically significant inhibition of growth rate and yield was found at all test concentrations. However, growth rate inhibition was considered to be biologically not relevant at 0.35, 0.51 and 0.90 mg/L, where the observed inhibition was below 10%. The NOEC based on biological relevance was thus set at 0.90 mg/L. Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to a loading rate of 1.0 mg/L when compared to the control.
Results with reference substance (positive control):
Potassium dichromate inhibited growth rate of this fresh water algae species at all concentrations tested.
The EC50 for growth rate inhibition (72h-ERC50) was 1.05 mg/L with a 95% confidence interval ranging from 1.04 to 1.06 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.6 mg/L. Hence, the 72h-ERC50 for the algal culture tested corresponds with this range. The EC50 for yield inhibition (72h-EYC50) was 0.295 mg/L with a 95% confidence interval ranging from 0.292 to 0.298 mg/L.
Reported statistics and error estimates:
For determination of the NOEC and the ECx the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Step-down Jonckheere-Terpstra Test Procedure, α=0.05, one-sided, smaller). Calculation of ECx-values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding TWA concentrations of the test item. 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, under the conditions of the present study with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), 1-(bis(2-(1,3dimethylbutylideneamino) ethyl)amino)-3-phenoxypropan-2-ol inhibited growth rate and yield of this fresh water algae species significantly at TWA concentrations of 0.35 mg/L and higher. The 72h-EC50 for growth rate inhibition (ERC50) was 2.4 mg/L with a 95% confidence interval ranging from 2.3 to 2.5 mg/L. The 72h-EC50 for yield inhibition (EYC50) was 1.2 mg/L with a 95% confidence interval ranging from 1.1 to 1.2 mg/L. The 72h-NOEC for growth rate inhibition was lower than 0.35 mg/L based on statistical significance and 0.90 mg/L based on biological relevance. The 72h-NOEC for yield inhibition was lower than 0.35 mg/L based on statistical significance and biological relevance.
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 toxic effects in Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) during an exposure period of 72 hours and, if possible, to determine the NOEC, EC10, EC20 and EC50 for both inhibition of growth rate and inhibition of yield. The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. 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 preceding range-finding test. Six replicates of exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to WAFs individually prepared at loading rates of 0.22, 0.46, 1.0, 2.2, 4.6 and 10 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, after 24 and 72 hours of exposure.  The actual test item concentrations based on the measurements of the ketone component were 0.57, 1.1, 1.2, 2.4, 4.6 and 9.5 mg/L at the WAFs prepared at 0.22, 0.46, 1.0, 2.2, 4.6 and 10 mg/L respectively, at the start of the test. At the end of the test, the two lowest test item concentrations were not detected. The 1.0 mg/L WAF decreased to 54% of initial at the end of the test, while higher concentrations remained stable (i.e. were at 85 – 100% of initial). The actual test item concentrations based on the measurements of the diamine component were 0.47, 0.61, 0.82, 1.9, 3.8 and 8.1 mg/L at the WAFs prepared at 0.22, 0.46, 1.0, 2.2, 4.6 and 10 mg/L respectively, at the start of the test. During the exposure period, the concentrations decreased to 24 - 92% of initial at the end of the test. Based on these results, effect parameters were expressed as TWA concentrations of the diamine component. However, for calculating the ECx-values the two lowest test item concentrations were removed from calculations. These analysed test item concentrations were assumed to not be test item related because they were comparable to the QC blank response. Inhibition of growth rate and yield increased with increasing concentrations of 1-(bis(2-(1,3dimethylbutylideneamino) ethyl)amino)-3-phenoxypropan-2-ol at all concentrations tested resulting in 100% inhibition at the highest concentration tested. The study met the acceptability criteria prescribed by the study plan and was considered valid.

The effect parameters obtained in this study are summarized in the table below.

Parameter (mg/L)                NOEC*       NOEC#       EC10       EC20       EC50

Growth rate Value                 <0.35       0.90             1.2            1.5               2.4  

lower 95%-cl                                                             1.1           1.4                 2.3  

upper 95%-cl                                                             1.4           1.7             2.5

Yield   Value                     <0.35           <0.35       0.63        0.78              1.2  

lower 95%-cl                                                           0.58         0.74              1.1  

upper 95%-cl                                                          0.67          0.83              1.2

cl – confidence limit, * - based on statistical significance, # - based on biological relevance