Ethylenebis(oxyethylene) bis[3-(5-tert-butyl-4-hydroxy-m-tolyl)propionate]

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1992-06-15 to 1992-09-16

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP-guideline study. Deviations to the updated OECD Guideline (adopted 2006): raw data re-evaluated.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

1987

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

yes

Remarks:

No detailed verification of the validity criteria in the original study report. The raw data has been re-evaluated and the validity criteria have been fulfilled.

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 2.0 g test substance and 18.0 g of a 0.4% alkylphenol-polyglycolether (ARKOPAL) were mixed together, by means of a marble-mill during about 96 hours. 1 ml of this blend were mixed and made up to 1000 ml with water achieving a concentration of 100 mg/l.
- Controls: Blank (water-composition according to the guideline 87/302/EEC) and vehicle (3.6 mg ARKOPAL/L).
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): alkylphenol-polyglycolether (ARKOPAL).

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green Algae.
- Strain: Scenedesmus subspicatus.
- Source (laboratory, culture collection): CIBA-GEIGY Ltd., testing facility.
- Laboratory culture: Yes.
- Method of cultivation: According to the guideline 87/302/EEC.
- Initial cell concentration: 9,900 cells/mL.
- Preculture: 3 days under test conditions.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

None

Test temperature:

23 ± 1°C.

pH:

7.7 to 9.9

Nominal and measured concentrations:

Nominal: 1.23, 3.7, 11, 33, and 100 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL Erlenmeyer flasks, stoppered with aluminium caps, on Lab-shaker, 50 mL test solution per flask.
- Temperature: 23 ± 1°C.
- Initial cells density: 9,900 cells/mL.
- No. of vessels per concentration (replicates): 3.
- No. of vessels per control (replicates): 6.
- No. of vessels per vehicle control (replicates): 3

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Composition according to the guideline 87/302/EEC.


OTHER TEST CONDITIONS
- Adjustment of pH: None.
- Photoperiod: Continuous illumination.
- Light intensity and quality: 134 µE/m2 sec ± 20% (approximately 8,000 lux); cold white fluorescent light.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: "TOA" cell counter.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Not exceeding 3.2.
- Test concentrations: Nominal: 1.23, 3.7, 11, 33, and 100 mg/l. A water and a vehicle control were tested in parallel.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Cell density after 72 hours: 2128000
- Exponential growth in the control (for algal test): Yes.
- Observation of abnormalities (for algal test): Not reported.
- Other: Inhibition was reported in the vehicle control group. pH values changed more than 1.5 units for the blank control and vehicle control groups.

Reported statistics and error estimates:

The EbC50 values were calculated according to BERKSON JASA 48 (1953), 569-599.  EC-values were graphically determined on gausso-logarithmic probability paper. 

Validity criteria requested by OECD TG 201 compared to the present study (re-evaluation of the raw data)

-      The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.

--> The biomass in the control cultures increased by a factor of 215 in the control and 199 in the vehicle control. The validity criterion is therefore fulfilled.

 

-      The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%.

Growth rates for each day derived by re-evaluation of the raw data.

	0-24	24-48	48-72	Mean	SD	CV [%]
Control	1.70	1.99	1.67	1.79	0.18	9.93
Vehicle control	1.59	2.07	1.62	1.76	0.27	15.27

 

--> The coefficients of variation do not exceed 35%. The validity criterion is fulfilled.

 

-      The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus.

--> The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was1.23% (SD 0.02). For the vehicle control the CV was1.20% (SD 0.02). The validity criterion is fulfilled.

The validity criteria are fulfilled and the study is regarded as valid.

Growth rate and yield

Additional data derived from the re-evaluation of the raw data of the study report.

Parameter	Control	Vehicle	1.23 mg/L	3.7 mg/L	11 mg/L	33 mg/L	100 mg/L
Yield after 72h	2118433	1956767	2276767	2471767	2450100	2491767	2543433
Inhibition relative to control [%] *	n/a	7.63	-7.47	-16.68	-15.66	-17.62	-20.06
Inhibition relative to vehicle [%] *	n/a	n/a	-16.35	-26.32	-25.21	-27.34	-29.98
Growth rate after 72h	1.79	1.76	1.81	1.84	1.84	1.84	1.85
Inhibition relative to control [%] *	n/a	1.46	-1.35	-2.86	-2.70	-3.04	-3.31
Inhibition relative to vehicle [%] *	n/a	n/a	-2.85	-4.38	-4.23	-4.57	-4.84

* Negative values indicate a stimulatory effect relative to the control/vehicle control.

Validity criteria fulfilled:

yes

Remarks:

Validity criteria are fulfilled; raw data re-evaluated

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Water Quality Measurements and Environmental Conditions
The pH of each control and test solution was measured at test initiation and at test completion. Conductivity was measured at test initiation only. Initial measurements at test initiation were measured in the bulk solutions, retained in a separate vessel to avoid contamination of the test solutions. Final pH measurements at test completion were measured in pooled solutions from the replicates of each control and test solution. Water quality was measured according to the Work Instruction for Operation and Maintenance of Thermo Orion A329 Multiparameter Meter (BBY2 WI-00026).
The temperature of the test chamber was monitored daily using a min/max thermometer, according to the Work Instruction for Temperature Monitoring (BBY WI-00024). Prior to test initiation, light intensity was measured according to the Work instruction for Operation and Maintenance of Light Meters (BBY2 WI-00016).

Test Observations
Observations of test solution appearance, including cloudiness or unusual appearance, were made and recorded during the enumeration process. Any unusual appearance of the algal cells, including observed differences in cell size, was noted if present.
Test treatments were enumerated by removing the test vessels from the incubation chamber and shielded from light (covered with black plastic). Replicate cell counts were performed on aliquots from each flask using the Cellometer X4HM Auto counter. The Cellometer X4HM Auto counter was set to count fluorescent algae-PKS cell type, with dilution factor of 1. The cell density in each replicate was determined by counting a minimum of four aliquots from each test vessel.

Confirmatory Analysis
Confirmatory analysis of the test solutions was measured in aliquots of each control and each test item loading rate solution at test initiation and at test completion. Aliquots from test initiation were collected from the vessels used to prepare the bulk test solutions. Aliquots from test completion were collected from the supernatant of the centrifuged solutions. Approximately 40 mL of each test solution and each control was collected in pre-conditioned amber glass vials of appropriate size and stored refrigerated (2°C to 8°C) until analysis. Approximately 1L of nutrient media (control media) was also collected at test initiation.

Vehicle:

no

Details on test solutions:

Selection of Test Solution Loading Rates and Experimental Approach
A series of preliminary tests were conducted (non-GLP) to determine the necessary parameters for testing Ethylenebis(oxyethylene) bis[3-(5-tert-butyl-4-hydroxy-m-tolyl)propionate], which ultimately led to the use of water accommodated fractions (WAF) with a preconditioning rinse. The test concentrations in the definitive test were dilutions of a nominal loading rate of 100 mg/L (6.25, 12.5, 25, 50 mg/L) along with a negative control and a water accommodated fraction (WAF) control (dilution water only).

Preliminary WAF Study
A preliminary WAF study was conducted to determine the appropriate WAF stirring time, and the efficacy of preconditioning. WAFs were prepared at 100 mg/L.

Range Finding Test
A range finding test was conducted (non-GLP) using dilutions of a nominal loading rate of 0.2 mg test item/L (0, 0.0002, 0.002, 0.02 mg/L). As the test item is a mono-constituent substance with a water solubility of 104 µg/L, the test solutions were prepared as dilutions of a 48 hour, 0.2 mg/L WAF. The estimated EyLR50 for cell yield based on nominal loading rates for the preliminary test was >0.2 mg test item/L (95% Confidence Limits: N/A). The estimated ErLR50 for specific growth rate based on nominal loading rates for the preliminary test was >0.2 mg test item/L (95% Confidence Limits: N/A). The test solutions were observed for a Tyndall effect and no Tyndall effect was observed.

Additional WAF Preparations
Additional WAF preparations were conducted as the range finding test did not result in measurable concentrations of the test item. Solutions were prepared in the same manner as the range finding test, and analysed. Confirmatory chemistry indicated that no peak was present in any of the solutions.
Next, the WAF was tested to determine if preparing a WAF at 100 mg/L would result in different measured concentrations than a WAF prepared at 0.2 mg/L. As a result, the 100 mg/L WAF preparation had measureable concentrations of the test item. Although the measured concentration was higher than the test item solubility, it was concluded that the 100 mg/L WAF preparation was superior to the 0.2 mg/L WAF preparation.
Moving forward, the WAFs were tested for media interference. 100 mg/L WAFs were prepared in media and deionised water (DI) to determine if the media was interfering with the test item solubility. Samples were collected in both 40 mL bottles and 100 mL bottles and analysed in duplicate. The results indicated that the media did not appear to be causing interferences with the test item, however samples collected in 40 mL bottles resulted in higher test item concentrations.

Preconditioning WAFs, Sample Homogeneity, and Sample Stability
WAFs were prepared to determine the best method for preconditioning test vessels and subsample bottles, as well as the best method for subsampling. A 100 mg/L WAF was prepared and sample bottles were rinsed with the WAF solution for varying repetitions. Subsample volumes were also tested to determine if homogeneity issues could be attributed to a lack of headspace and ease of mixing the solution in the subsample bottles. From the results, it was determined that 1 preconditioning rinse would be used for the test, and the sample bottles would be filled to include a small amount of headspace. This allows for ease of mixing when conducting confirmatory analysis. An additional sample was collected in which acetonitrile (ACN) was added directly to the sample bottle prior to analysis to determine the concentration of test item without adhesion to the sample bottle.
Variability between solutions and duplicate analysis continued to be a concern. It was discussed if free particles of the test item were present in the WAF solutions. Thus, 100 mg/L WSFs were prepared using filtration as the separation technique. Glass fiber filters (0.5 µm) were preconditioned with test solutions, by filtering a volume of the WAF solution and discarding the filtrate prior to filtering the solution for analysis. Centrifugation was not employed as the large volume of test solutions required limited the ability to centrifuge. WSFs were prepared in both beakers and aspirator bottles. The resulting chemistry analysis indicated that filtration removed all test item and peaks were not observed during analysis. As filtration was not a viable separation technique, WAFs were prepared, and test solutions were observed for the Tyndall effect.
Storage stability of the samples was also investigated. The preliminary WAF study samples were re-analysed on two separate occasions.

Preliminary Testing Conclusion
In conclusion, the definitive test was initiated using dilutions of a nominal test item loading rate of 100 mg test item/L (6.25, 12.5, 25, 50) along with a negative control and a water accommodated fraction (WAF) control (dilution water only). The test vessels and subsample bottles were pre-conditioned by thoroughly rinsing with the appropriate test solution. WAFs were observed for the presence of the Tyndall effect, and subsamples were analysed as soon as possible, with a maximum holding period of 7 days.

Test Solution Preparation
The test item is a difficult item as defined by the OECD Guideline 23 (2000) because it is poorly soluble in water. The test item is a mono-constituent substance; with a water solubility of 104 µg/L. In order to maximise the amount of test item in solution, a 100 mg/L solution was prepared as a water accommodated fraction (WAF). Test solutions were prepared as dilutions the 100 mg/L WAF solution and followed the methodology described below. The test vessels, holding vessels, and subsample vials used in the study were pre-conditioned by rinsing once with the test solution prior to use.
To prepare the 100 mg/L WAF, the test item was weighed on a watchglass and added directly to ~1000 mL (i.e., approximately half volume) of nutrient media (dilution water) in a ~2 L glass vessel, and the volume topped up to 2 L with the nutrient media. The WAF control was prepared in a similar manner; however, no test item was added. Each WAF was stirred using a magnetic stir bar on a stir plate to create a vortex (~10%) and covered (e.g., sterile aluminum foil) to reduce contaminants. Stirring occurred for approximately 44 hours, followed by an approximately 4 hour settling period.
WAF solutions were removed from the WAF vessel and collected in a glass holding vessel (1000 mL glass beaker) by siphoning from the middle of the water column using Teflon tubing, discarding the first ~100 mL into an appropriate container.
The test item was handled according to the Work Instruction for Management of Test and Reference Items for GXP Studies (BBY1 WI-00008). All chemicals were weighed using analytical balances, which were operated according to the procedures outlined in the Work Instruction for Corporate Procedure for Balance Calibrations and Verifications (COR WI-00015). All containers and apparatus used were new or thoroughly cleaned according to the Work Instruction for Cleaning of Laboratory Ware (BBY WI-00001).
The test solutions and test maintenance was performed in a laminar flow hood according to the Safe Work Procedure for Laminar Flow Hoods and Biological Safety Cabinets (MEHSSWP-00031). All equipment and glassware was sterilized according to the Work Instruction for the Operation and Maintenance of Sterilizers (BBY2 WI-00019) or by using a 70% ethanol solution.

Test organisms (species):

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

Details on test organisms:

Test Organism
Pseudokirchneriella subcapitata, (strain CPCC 37, also known as Raphidocelis subcapitata), was obtained from the Canadian Phycological Culture Centre (formerly known as University of Toronto Culture Collection) as a sterile liquid starter culture (CP180214). The starter culture was used to initiate new algae cultures. The algae were cultured in 250 mL glass Erlenmeyer flasks, using nutrient mediumhardness
asdfasdfasdfasdf, and testing was based on the procedures in the Standard Operating Procedure OECD 201: Alga, Growth Inhibition (BBY2SOP-00016).

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22°C – 23°C

pH:

initial (0h): 7.5 - 7.6
final (72h): 8.1 - 8.3

Dissolved oxygen:

n/a

Salinity:

n/a

Conductivity:

91-92 µS/cm

Nominal and measured concentrations:

Dilutions of a nominal loading rate of 100 mg test item/L (6.25, 12.5, 25, 50 mg/L)
A 100 mg/L nominal loading rate resulted in a measured initial concentration of 6.66 mg/L (above quantitation level and above water solubility of the test item)

Details on test conditions:

Test Conditions
Test type: Static
Duration: 72 hours ± 1 hour
Test organism species: Pseudokirchneriella subcapitata (also known as Raphidocelis subcapitata)
Age of test organisms at test initiation: 3 days
Source of test organisms: In-house culture; original culture was obtained from Canadian Phycological Culture Center (Waterloo, ON) on February 14, 2018 (CP180214)
Test organism identification: 2018Oct20A
Culture and testing medium: Sterile nutrient growth media
Incubator Temperature: 22°C – 23°C
Photo-period: Continuous (24-h light)
Light intensity: 5678 – 6334 lux
Test vessels: 250 mL Erlenmeyer flasks covered with a foam stopper, mixed on a horizontal shaker at approximately 75 rpm
Test volume: 50 mL
pH of test solutions: 7.5 – 8.3
Concentration of cells inoculated per test vessel: 0.99 x 104 cells/mL
Replicates: Six replicates for each control; 3 replicates for each nominal test item loading rate
Controls: Negative control and WAF control (dilution water only)
Nominal concentrations of the Test Item: 6.25, 12.5, 25, 50, and 100 mg/L
Water quality measurements: pH at test initiation and at test completion; conductivity at test initiation; temperature daily in test chamber
Measurement endpoints: Cell Yield and Specific Growth Rate (SGR); ELR50/EC50, ELR20/EC20, ELR10/EC10, NOELR/NOEC, LOELR/LOEC

Nutrient growth medium was used for culturing P. subcapitata, and for testing. The pH of the nutrient medium was 7.6 and no additional pH adjustment was needed. The media was filter sterilised through a 0.2 µm filter prior to use. The nutrient medium was prepared according to the recipe in the Standard Operating Procedure for Pseudokirchneriella subcapitata 72H Growth Inhibition Test (BBY2SOP-00006).

Recipe for Nutrient Growth Medium (amounts given in µg/L)
NaNO3: 25,500
MgCl2*6H2O: 10,000
CaCl2*2H2O: 4,420
H3BO3: 186
MnCl2*4H2O: 416
ZnCl2: 3.28
CoCl2*6H2O: 1.43
CuCl2*2H2O: 0.012
Na2MoO4*2H2O: 7.26
FeCl3*6H2O: 160
Na2EDTA*2H20: 300
MgSO4*7H20: 14,700
K2HPO4: 1,044
NaHCO3: 15,000

Reference substance (positive control):

yes

Remarks:

CAS# 7446-20-0 conducted twice annually

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 6.66 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Remarks on result:

other: measured

Remarks:

above quantitation level; estimated of the measure concentration

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 6.66 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Remarks on result:

other: measured

Remarks:

above quantitation level; estimated of the measure concentration

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

>= 6.66 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Remarks on result:

other: measured

Remarks:

above quantitation level; estimated of the measure concentration

Details on results:

At initiation of the WAFs, the 100 mg/L WAF appeared clear and colourless with test item spinning in the vortex. The WAF control was clear and colourless. After ~44 hours of mixing, the 100 mg/L WAF appeared clear and colourless with test item visible in the water column, on the water’s surface, on the bottom of the WAF vessel, and on the sides of the WAF vessel near the water’s surface. The WAF control appeared clear and colourless. After a 4 hour settling period, the WAF was siphoned, and test solutions were prepared. At test initiation, the negative control, WAF control, and all test item loading rates appeared clear and colourless without the presence of a Tyndall effect. All test solutions remained consistently clear and colourless until the ~24 hour observation time point. At the ~48 hour observation time point, the negative control, WAF control, and all test item loading rate solutions appeared clear and pale green. At test completion (72 ± 1 hour), all test solutions appeared green.
The 72 hour ELR50, ELR20, and ELR10 with 95% confidence limits (based on nominal loading rates) for cell yield and specific growth rate were calculated using Linear Interpolation (ICPIN). The NOELR (No Observed Effect Loading Rate) and LOELR (Lowest Observed Effect Loading Rate) values for cell yield and SGR were calculated using Dunnett Multiple Comparison Test. All statistical analyses and comparisons were performed against the WAF control. The statistically significant difference between the negative control and the WAF control was assessed using Equal Variance t-Two-Sample Test. Statistical analysis could not be conducted on the initial measured concentrations due to the non-linear test item concentrations. As there was no effect observed in the 100 mg/L nominal loading rate, it can be concluded that there was no effect observed in the 6.66 mg/L initial measured concentration which is far above the actual water solubility of the compound.

Results with reference substance (positive control):

The 72 hour EC50 and 95% confidence limits based on cell yield for the reference item was within the acceptable range (+- 2 SD) of previous tests conducted in this laboratory and indicates the test organism responded normally to the reference item.
Reference Item Test
Test Initiation Date: 2018 Nov 12
72h EC50 [mg/L Zn2+]: 0.073
95% Confidence Limits [mg/L]: 0.059, 0.081
+- 2SD of Previous Mean [mg/L]: 0.034, 0.106
Calculation Method: Linear Interpolation (ICPIN)

Validity criteria fulfilled:

yes

Conclusions:

Test Validity Criteria Results
The negative control was used for the assessment of test validity criteria.

Summary of Test Validity Criteria Results
The biomass in the control cultures must increase by a factor of at least 16 over the course of the 72 hour test period. --> The biomass in the control increased by a factor of 79
The coefficient of variation of average specific growth rates over the test period in replicate control cultures must not exceed 7%. --> The %CV of mean SGR in the control was 3.05%
The mean coefficient of variation (%CV) for the specific growth rates of each time period (days 0-1, 1-2, and 2-3) in the control cultures must not exceed 35%. --> The highest mean %CV in the control was 25.47%

Description of key information

There is a high probability that the product is not acutely harmful to aquatic algae.

Additional information

To determine the acute toxicity towards aquatic algae two experimental studies are available. A non-GLP study according to 87/302/EEC page 89-94 Algal growth inhibition test (similar to OECD guideline 201) was conducted in 1992. The raw data of this study was re-evaluated in 2017 to confirm the validity of the study. All validity criteria listed in paragraph 11 of OECD TG 201 are met and the study is regarded as reliable. No toxic effects (yield and growth rate) occur within the range of water solubility after 72 h up to the highest tested concentration (100 mg/L nominal).

A second study was requested in ECHA’s compliance check with the decision number CCH-D-2114381462-49-01/F. This study was conducted according to OECD TG 201 under consideration of GLP and OECD GD 23 for difficult to test substances. A loading rate of 100 mg/L did not reveal any toxic effects and the ErC50 and ErC10 values were determined to be >100 mg/L (nominal), >6.6 mg/L (initial measured). The NOErC was determined to be >= 100 mg/L (nominal) and >=6.6 mg/L (initial measured). The result confirmed the already existing data from 1992.

In conclusion, it can be assumed that the substance is not harmful to aquatic algae.