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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Test dates were from 15 to 18 January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 26D647
- Purity test date: October 5, 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient
- Appearance: liquid

Analytical monitoring:
yes
Details on sampling:
Samples were collected from the bulk test solutions at test initiation, from replicate vessels (pooled) at approximately 24 and 48 hours of exposure and at exposure termination. Blank replicates were sampled separately at approximately 24 and 48 hours and at exposure termination. All test solutions were analyzed within 24 hours of preparation.
Vehicle:
no
Details on test solutions:
Results of the non-GLP range-finding test and preliminary non-GLP methodology work indicated that the test material is expected to be completely soluble in the algal medium at the selected test concentrations for the study. A 1000 mg 2-(Bis(2-ethylhexyl)amino)ethanol/L nominally-dosed saturated solution was prepared via direct addition of the test material to algal medium. The solution was cloudy with clear liquid/droplets at the surface and throughout. The solution was then sonicated for 30 minutes via water bath sonication. Following sonication, the solution was cloudy with clear liquid/droplets at the surface. Approximately 1450 mL of the nominally-dosed saturated solution was then transferred to a glass beaker via filtering
through a Polycap 36 AS Whatman 0.45 µm nylon filter creating a bulk test solution equal to the highest test concentration (100% saturated solution). Remaining bulk test solutions were prepared as dilutions (in AAP medium) of the 100% saturated solution.

The bulk algal medium control consisted of algal medium without the addition of the test material. Prepared bulk test solutions were apportioned into individual test vessels. The test solutions were utilized on the same day as preparation; thus, assessment of stability of the test solutions was not required. The dispersal of the test material in the surrounding medium was considered to represent the most probable route of exposure in the environment.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Raphidocelis subcapitata from in-house cultures was initially obtained from the University of Texas at Austin Culture Collection (UTEX1648; lot # 060215) in June 2015. This species is widely accepted and recommended for toxicity testing by the test guidelines. Stock cultures of this organism were maintained axenically by periodic transfer into sterile medium. Algae were cultured under continuous illumination of approximately 5,200 ± 520 lux at a temperature of 23 ± 2ºC.
The algal inoculum for the test was prepared from a 3-day old stock culture. A Coulter Multisizer 3 (Beckman Coulter, Brea, California) was used to determine the cell density of the stock culture. This evaluation determined that a 0.190 mL aliquot of the culture was required to inoculate each test vessel at an initial cell density of approximately 10,000 cells/mL.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
23°C
pH:
7.3 -7.7
Salinity:
NA
Nominal and measured concentrations:
0 (AAP medium control), 1.00, 3.10, 9.80, 31.0 and 100% of a 1000 mg 2-[Bis(2-ethylhexyl)amino]ethanol/L nominally-dosed saturated solution
Details on test conditions:
The algae was cultured in freshwater algal nutrient medium (i.e., AAP medium), prepared with sterile deionized water and reagent grade chemicals. The water source for the deionized water system was Lake Huron water supplied to The Dow Chemical Company by the City of Midland Water Treatment Plant. The base water (laboratory dilution water (LDW)) used to prepare the medium was passed through a series of activated carbon, (two) deionization polymer (US Filter Mixed Bed, Type 1), and a final filtration unit, prior to collection and autoclaving in clean glass containers. The base water used to prepare the medium is analyzed periodically to verify that no contaminants are present at levels that may interfere with the test results.

Test vessels were sterilized 250-mL Erlenmeyer flasks with foam stoppers each containing 50 mL test medium. Each flask was uniquely labeled (i.e., study, replicate, test level) for identification purposes.

The definitive test was conducted under static conditions for approximately 72 hours from 15 to 18 January 2018. Three replicate test vessels were prepared per test level. Six replicates were prepared for the AAP medium control. Each replicate contained
50 mL of the appropriate test solution and was inoculated with approximately 10,000 cells/mL. An additional replicate at each test level and AAP medium control was prepared but not inoculated with algae to serve as a counting blank. These blanks were used to correct the daily counts for the potential interference of the test material and to monitor pH without the algal biomass. At test initiation and following sampling for cell densities at approximately 24 and 48 hours, the replicate test vessels were placed in a walk-in environmental chamber (Lab-Line Environmental Chamber, Lab-Line Inc., Melrose, Illinois) on a shaker table (set at approximately 100 rpm) according to a computer-generated randomization. The target test temperature was 23 ± 2ºC. The photoperiod was set at 24 hours of continuous light with a target light intensity of 5,200 ± 780 lux. The pH was measured from bulk test solutions at 0 hours and from blanks and pooled replicate samples of each test level and AAP medium control at 72 hours. Pooled samples were prepared by withdrawing and combining approximately 2.5-mL volumes from each inoculated replicate at each test level and AAP medium control. Temperature was continuously monitored with a minimum/maximum thermometer placed in a representative vessel containing deionized water located in the test area. At test initiation, light intensity was measured at each position where replicate vessels were placed during the exposure

Algal cell densities of the initial inoculum and test solutions were determined by electronic particle counting using a Coulter Multisizer 3 (Beckman Coulter, Brea, California) fitted with a 100-µm aperture tube. Total cell counts (average of two cell counts per replicate test vessel) were determined at approximately 24, 48 and 72 hours (± 1 hour from test initiation). Cells were cumulatively counted at a lower threshold equivalent spherical diameter of approximately 2.6 μm to a higher threshold equivalent spherical diameter of approximately 8.7 μm. The cell count values for the blank replicates were used to correct for background in daily calculations. In addition, morphological observations were done at exposure termination on a composited sample of the inoculated replicates at each test level. The cells were observed under a microscope (Olympus BH Microscope, (Olympus Corporation, Tokyo, Japan); 20x or 40x objective lens; WF10x eyepiece; 1.25x Dual Observation Deck).
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.073 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.029 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.017 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.018 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
other: Yield
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.166 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.127 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.067 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.018 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Details on results:
None of the analyses of the medium control exhibited a concentration exceeding the lower limit of quantitation (LLQ) equivalent to 0.00200 mg 2-(Bis(2-ethylhexyl)amino)ethanol/L. At test initiation (0 hours), measured 2-(Bis(2-ethylhexyl)amino)ethanol concentrations of the bulk test solutions (without algae) ranged from 0.00275 to 0.277 mg 2-(Bis(2-ethylhexyl)amino)ethanol/L. At 24 hours, the analysis of pooled replicate test solutions (with algae) yielded 2-(Bis(2-ethylhexyl)amino)ethanol concentrations ranging from
Geometric mean measured concentrations of the bulk test solutions (without algae) at 0 hours and pooled replicate solutions (with algae) at 24, 48 and 72 hours were 0.00129, 0.00424, 0.0183, 0.0510 and 0.166 mg 2-(Bis(2-ethylhexyl)amino)ethanol/L and were used in reporting study endpoints. Similarly, the geometric mean measured concentrations of the bulk test solutions (without algae) at 0 hours and the blank replicate solutions (without algae) at 24, 48 and 72 hours were 0.00129, 0.00393, 0.0168, 0.0487 and 0.159 mg 2-(Bis(2-ethylhexyl)amino)ethanol/L. Where measured concentrations were
Temperature during the exposure period was 23ºC. Light intensity ranged from 4890-5850 lux. The pH of bulk test solutions (without algae) were 7.5 at test initiation. At exposure termination, the pH was 7.7 in pooled replicates (with algae) and 7.3 in blank replicates (without algae).

All cell yield data were normally distributed and homogeneous (Shapiro-Wilk Test, p > 0.01 and Levene’s Test, p > 0.01). Mean yields at 72 hours were 272.6, 256.4, 230.2, 257.4, 169.5 and 62.63 (x104) cells/ml for the AAP medium control, 0.00129, 0.00424, 0.0183, 0.0510 and 0.166 mg 2-(Bis(2-ethylhexyl)amino)ethanol/L test levels, respectively. At 72 hours, the mean inhibition response relative to the AAP medium control ranged from 6 to 77% inhibition of yield. Based on statistical analysis of the results (Dunnett’s test, α = 0.05), cell yield at the 0.0510 and 0.166 mg 2-(Bis(2-ethylhexyl)amino)ethanol/L test levels were significantly different from the AAP medium control. Thus, the 72-hour NOEC for yield is reported as 0.0183 mg 2-(Bis(2-ethylhexyl)amino)ethanol/L. The statistically calculated 72-hour EyC50 (95% confidence intervals) is 0.0734 (0.0660 – 0.0808) mg 2-(Bis(2-ethylhexyl)amino)ethanol/L. The statistically calculated 72-hour EyC20 (95% confidence intervals) is 0.0292 (0.0235 – 0.0350) mg 2-(Bis(2-ethylhexyl)amino)ethanol/L and the statistically calculated 72-hour EyC10 (95% confidence intervals) is 0.0171 (0.0124 – 0.0217) mg 2-(Bis(2-ethylhexyl)amino)ethanol/L.

All growth rate data were normally distributed and homogeneous (Shapiro-Wilk Test, p > 0.01 and Levene’s Test, p > 0.01). Mean specific growth rates from 0 to 72 hours were 1.868, 1.850, 1.813, 1.851, 1.711 and 1.383 (day-1) for the AAP medium control, 0.00129, 0.00424, 0.0183, 0.0510 and 0.166 mg 2-(Bis(2-ethylhexyl)amino)ethanol/L test levels, respectively. From 0 to 72 hours, mean inhibition response relative to the AAP medium control ranged from 1 to 26% of the mean specific growth rate. Based on statistical analysis (Dunnett’s test, α = 0.05) of the results, mean specific growth rate at the 0.0510 and 0.166 mg 2-(Bis(2-ethylhexyl)amino)ethanol/L test levels were significantly different from the AAP medium control. Thus, the 72-hour NOEC for growth rate is reported as 0.0183 mg 2-(Bis(2-ethylhexyl)amino)ethanol/L. The statistically calculated 72-hour ErC50 (95% confidence intervals) is >0.166 mg 2-(Bis(2-ethylhexyl)amino)ethanol/L (highest concentration tested). The statistically calculated 72-hour ErC20 (95% confidence intervals) is 0.127 (0.113 – 0.143) mg 2-(Bis(2-ethylhexyl)amino)ethanol/L and the statistically calculated
72-hour ErC10 (95% confidence intervals) is 0.0666 (0.0533 – 0.0832) mg 2-(Bis(2-ethylhexyl)amino)ethanol/L.

At test termination, microscopic evaluation of algal cells at each test level, including the AAP medium control, revealed no abnormal observations.
Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity values for Raphidocelis subcapitata exposed to 2-[Bis(2-ethylhexyl)amino]ethanol over a 72-hour static exposure period and based on geometric mean measured concentrations were as follows:
Cell yield
72-hour EyC50: 0.0734 mg/L (95% C.I. = 0.0660 – 0.0808 mg/L)
72-hour EyC20: 0.0292 mg/L (95% C.I. = 0.0235 – 0.0350 mg/L)
72-hour EyC10: 0.0171 mg/L (95% C.I. = 0.0124 – 0.0217 mg/L)
72-hour NOEC: 0.0183 mg/L
Growth rate
72-hour ErC50: >0.166 mg/L (highest concentration tested)
72-hour ErC20: 0.127 mg/L (95% C.I. = 0.113 – 0.143 mg/L)
72-hour ErC10: 0.0666 mg/L (95% C.I. = 0.0533 – 0.0832 mg/L)
72-hour NOEC: 0.0183 mg/L
Executive summary:

The purpose of this study was to assess the potential effects of 2-[Bis(2-ethylhexyl)amino]ethanol tothe freshwater green alga, Raphidocelis subcapitata. The study was performed for 72 hours with target nominal test concentrations of 0 (AAP medium control), 1.00, 3.10, 9.80, 31.0 and 100% of a 1000 mg 2-[Bis(2-ethylhexyl)amino]ethanol/L (AAP medium) nominally-dosed saturated solution. Cell density was determined at approximately 24, 48 and 72 hours (±1 hour from test initiation). Temperatures during the exposure were 23°C. The pH ranged from 7.3-7.7 and the light intensity ranged from
4890-5850 lux.

Test solutions were analyzed for 2-[Bis(2-ethylhexyl)amino]ethanol concentrations at test initiation, following 24 and 48 hours of exposure and at exposure termination by high performance liquid chromatography/mass spectrometry (HPLC/MS-MS)None of the analyses of the AAP medium control exhibited a concentration exceeding the lowest limit of quantitation (LLQ) equivalent to 0.00200 mg 2-[Bis(2-ethylhexyl)amino]ethanol/L. Measured concentrations of2-[Bis(2-ethylhexyl)amino]ethanolranged from <LLQ to 0.277 mg 2-[Bis(2-ethylhexyl)amino]ethanol/L over the course of the exposure period. The resulting geometric mean measured test concentrations were 0.00129, 0.00424, 0.0183, 0.0510 and 0.166 mg 2-[Bis(2-ethylhexyl)amino]ethanol/L. Where measured concentrations were <LLQ (excluding medium control analyses), a concentration value of ½ LLQ was utilized for determining mean measured concentrations.

The acute toxicity values forRaphidocelis subcapitataexposed to 2-[Bis(2-ethylhexyl)amino]ethanol over a 72-hour static exposure period and based on geometric mean measured concentrations were as follows:

  • Cell yield

72-hour EyC50: 0.0734 mg/L (95% C.I. = 0.0660 – 0.0808 mg/L)

72-hour EyC20:0.0292 mg/L (95% C.I. = 0.0235 – 0.0350 mg/L)

72-hour EyC10: 0.0171 mg/L (95% C.I. = 0.0124 – 0.0217 mg/L)

72-hour NOEC: 0.0183 mg/L

  • Growth rate

72-hour ErC50: >0.166 mg/L (highest concentration tested)

72-hour ErC20: 0.127 mg/L (95% C.I. = 0.113 – 0.143 mg/L)

72-hour ErC10: 0.0666 mg/L (95% C.I. = 0.0533 – 0.0832 mg/L)

72-hour NOEC: 0.0183 mg/L

Description of key information

The acute toxicity values forRaphidocelis subcapitata exposed to 2-[Bis(2-ethylhexyl)amino]ethanol over a 72-hour static exposure period and based on geometric mean measured concentrations were as follows:

  • Growth rate

72-hour ErC50: >0.166 mg/L (highest concentration tested)

72-hour NOEC: 0.0183 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
0.166 mg/L
EC10 or NOEC for freshwater algae:
0.018 mg/L

Additional information