Bis[4-(tert-butyl)benzoato-O]hydroxyaluminium

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:

2018-07-15 - 2018-07-25 (experimental phase)

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:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

OECD Guidelines for the Testing of Chemicals, Section 2, No. 201: "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", adopted March 23, 2006, corrected July 28, 2011

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Commission Regulation (EC) No 761/2009, Annex, Part C, C.3.: "Freshwater Algae and Cyanobacteria, Growth Inhibition Test", Official Journal of the European Union (EN), dated August 24, 2009

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures", December 15, 2000

Deviations:

not applicable

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Bis[4-(tert-butyl)benzoato-O]hydroxyaluminium

EC Number:

236-109-7

EC Name:

Bis[4-(tert-butyl)benzoato-O]hydroxyaluminium

Cas Number:

13170-05-3

Molecular formula:

C22H27AlO5

IUPAC Name:

aluminum hydroxide bis(4-tert-butylbenzoate)

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: filtrate of nominal 100 mg/L and dilutions of 1:2, 1:4, 1:8 and 1:16 of this filtrate, corresponding to nominal loading rates of 100, 32, 10, 3.2 and 1.050, 25, 12.5 and 6.25 mg test item/L (spacing factor 2), and a control.
- Sampling method: The samples were taken from the biological phase of the study. Duplicate samples from the freshly prepared test media (containing algae) of all test concentrations and from the control were taken at the start of the test.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of all test concentrations and the control (containing algae) were taken at the end of the test (after the 72 hours test period) by pouring together the contents of the test beakers of each treatment.
- Sample storage conditions before analysis: All samples were stored in a refrigerator (4 ± 4 °C), protected from light until analysis was performed.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was not well soluble in test water. In this study, only the inhibitory effects of dissolved test item was tested and thus no concentrations above the solubility limit of the test item in test water were tested. Therefore, a supersaturated stock solution of nominal 100 mg test item/L was prepared by suspending 200 mg test item in 2000 mL test water for preparing the test concentrations. The stock suspension was overhead shaken for 48 hours at room temperature in the dark to dissolve as much test item as possible. Then, non-dissolved fractions of the test item were separated from the test medium by membrane filtration (0.45 µm cellulose acetate filter). The solution with dissolved test item was used as the test medium of the highest test concentration and to prepare the desired 1:2, 1:4, 1:8 and 1:16 dilutions.
The test media were prepared just before introduction of the algae (= start of the test).
- Controls: In the control, test water was used without addition of the test item.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (KORSHIKOV), formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV).
- Strain: Strain No. 61.81 SAG
- Source (laboratory, culture collection): The algae were cultivated in the laboratories of ibacon. The algae were originally supplied by the „Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen", 37073 Göttingen, Germany.
- Method of cultivation: The algae were cultivated under standardised conditions according to the test guidelines.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L) as CaCO3

Test temperature:

22.1 to 23.9 °C

pH:

7.9 to 8.2 at test start;
7.9 to 9.2 at test end

Nominal and measured concentrations:

Due to the poor water solubility of the test item, a filtrate of nominal 100 mg/L and dilutions of 1:2, 1:4, 1:8 and 1:16 of this filtrate were tested.
Nominal loading rates: 100, 50, 25, 12.5 and 6.25 mg test item/L
Arithmetic mean measured concentrations (based on TOC-measurement and specified carbon content of the test item): 25.9, 13.1, 6.70, 3.29 and 1.64 mg test item/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Erlenmeyer flasks of 200 mL volume with approximately 100 mL of test medium
- Aeration: no
- Initial cells density: 5000 algal cells per mL test medium
- Control end cells density: 111.817 [x 10000 cells/mL]
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Water (OECD Medium). Analytical grade salts and additives were added in deionised water at defined nominal concentrations (see "Any other information on materials and methods incl. tables").
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: The pH of the test water was adjusted with 1 M NaOH from pH 7.2 to pH 8.1.
- Photoperiod: Continuous illumination
- Light intensity and quality: Mean light intensity: 5735 lux (range: 5010 to 6240 lux). The light intensity was measured once during the test at 0 positions distributed over the experimental area at the surface of the test media.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The inhibition of algal growth was determined from the groth parameter yield (y) and average specific growth rate (µ) after 24, 48 and 72 hours of exposure. The yield is defined as the actual number of cells per volume of medium (cells/mL) at the end of exposure minus the cell number at the start of the study. The average specific growth rate is defined as the increase of cell density per time unit.
- Determination of cell concentrations: The cell density on each observation time was determined by spectrophotometric measurement. Therefore, defined volumes of the algal suspensions from all replicates and from the blanks were sampled after 24, 48 and 72 hours of exposure, and were not replaced. The algal cell densities were calculated by subtracting the absorption of the blanks, from each of the measured absorption of the test media (with algae).
Based on the counted cell densities and the absorption from an algal suspension and its dilutions, a linear regression was performed for the calculation of the cell densities of the replicates during the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: Pre-experiments were performed to determine a suitable concentration range and to establish suitable methods to prepare the test solutions.
- Test concentrations: Due to the poor water solubility of the test item, a filtrate of nominal 100 mg/L and dilutions of 1:10, 1:100 and 1:1000 of this filtrate were tested in the range finding study, corresponding to nominal loading rates of 100, 10, 1.0 and 0.10 mg test item/L. In the definitive study a filtrate of nominal 100 mg/L and dilutions of 1:2, 1:4, 1:8 and 1:16 were tested, corresponding to nominal loading rates of 100, 50, 25, 12.5 and 6.25 mg test item/L.
- Results used to determine the conditions for the definitive study: In the range finding study an inhibition of 93.0 % was determined for the parameter yield and an inhibition of 50.0 % was determined for the parameter growth rate at the highest nominal loading rate of 100 mg/L. No inhibition was observed at the lower nominal loading rates of 10, 1.0 and 0.1 mg/L for both growth parameter.

Reference substance (positive control):

yes

Remarks:

The reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions. The most recent test with Pseudokirchneriella subcapitata was performed in January 2018.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

70.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% conf. interval: 64.0 - 78.1 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% conf. interval: not determinable

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

58.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% conf. interval: 52.7 - 65.0 mg/L

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

77.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% conf. interval: 72.6 - 83.2 mg/L

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

52.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% conf. interval: 46.7 - 59.5 mg/L

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

67.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% conf. interval: 61.1 - 75.2 mg/L

Effect concentrations 7

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 8

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 9

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 10

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 11

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

18.4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% conf. interval: 16.7 - 20.3 mg/L

Effect concentrations 12

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 25.9 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% conf. interval: 25.8 - 26.5 mg/L

Effect concentrations 13

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

15.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% conf. interval: 13.8 - 16.9 mg/L

Effect concentrations 14

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

20.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% conf. interval: 18.9 - 21.6 mg/L

Effect concentrations 15

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

13.8 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% conf. interval: 12.3 - 15.6 mg/L

Effect concentrations 16

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

17.7 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% conf. interval: 16.0 - 19.6 mg/L

Effect concentrations 17

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

13.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 18

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

25.9 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 19

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

13.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 20

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

25.9 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing up to a nominal loading rate of 100 mg test item/L and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to this test concentration, the highest concentration tested.
- Any stimulation of growth found in any treatment: Within the lower loading rates of nominal 25, 12.5 and 6.25 mg/L a slight stimulation of growth was observed. As this stimulation is not significant compared to the control and not dose related, it is considered to be the reason of natuaral biological variation of algal growth.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: In the most recent test with the reference item Potassium dichromate, performed in January 2018, the EC50 for yield was 0.402 mg test item/L, the EC50 for growth rate was 0.963 mg test item/L and the EC50 for biomass was 0.414 mg test item/L.
- Other: In the reference test with the reference item Potassium dichromate, performed in July 2017, the EC50 for yield was 0.415 mg test item/L, the EC50 for growth rate was 1.02 mg test item/L and the EC50 for biomass was 0.455 mg test item/L, showing that the sensitivity of the test organism did not change significantly over time.

Reported statistics and error estimates:

Based on the calculated cell densities, the 72 hours ErL50 and the 72 hours EyL50, the corresponding EL20 and EL10 values and where possible their 95 %-confidence limits were calculated by Probit analysis.
For the determination of the 72 hours LOEL and the 72 hours NOEL, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Williams t-test (yield and growth rate).
The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat(R) Solutions GmbH.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The study was conducted under GLP according to OECD guideline 201 and EU Method C.3 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or deviations from the guidelines, the validity criteria were met. Hence, the results can be considered as reliable to assess the toxicity of bis[4-(tert-butyl)benzoato-O]hydroxyaluminium towards algae.

The influence of bis[4-(tert-butyl)benzoato-O]hydroxyaluminium on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test.
The 72 -hour EyL50 was calculated to be 70.7 mg test item/L and the 72 -hour ErL50 value was calculated to be > 100 mg test item/L, corresponding to mean measured concentrations of 18.4 and > 25.9 mg test item/L, respectively. The 72-hour NOEyL was determined to be 50 mg test item/L and the associated 72-hour LOEyL was 100 mg test item/L, corresponding to mean measured concentrations of 13.1 and 25.9 mg test item/L, respectively. The 72-hour NOErL was determined to be 50 mg test item/L and the associated 72-hour LOErL was 100 mg test item/L, corresponding to mean measured concentrations of 13.1 and 25.9 mg test item/L, respectively.
The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to nominal loading rates since the maximal soluble test item concentration was applied to the test with the highest test concentration and the analytical determination confirmed the dilution factors of the lower test concentrations. Additionally the arithmetic mean measured test item concentrations were used for the result evaluation.
Based on the obtained results, bis[4-(tert-butyl)benzoato-O]hydroxyaluminium does not need to be classified as acutely toxic to the environment according to Regulation 1272/2008 and amendments. Furthermore, bis[4-(tert-butyl)benzoato-O]hydroxyaluminium does not need to be classified as chronic toxic to the environment according to Regulation 1272/2008 and amendments. Even if the 72 -hour ErL50 of > 100 mg test item/L, corresponding to an 72 -hour ErC50 of > 25.9 mg/L based on mean measured concentrations of the test item, is below the limit value for classification as aquatic chronic Cat. 3 (> 10 to

Executive summary:

The study was conducted under GLP according to OECD guideline 202 and EU Method C.2 on the registered substance itself.

The purpose of this test was to determine the inhibitory effect of the test item bis[4-(tert-butyl)benzoato-O]hydroxyaluminium on the growth of the freshwater green algae Pseudokirchneriella subcapitata. For this purpose, exponentially growing cultures of this unicellular green algal species were exposed to various concentrations of the test item under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations.

This study encompassed 6 treatment groups (5 dose rates of the test item and a control) with three replicates per test concentration and six replicates for the control. At test start 100 mL of the test concentrations were inoculated with nominal 5000 algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72 hours for determination of cell densities by spectrophotometrical measurement.

Due to the poor water solubility of the test item, a filtrate of nominal 100 mg/L and dilutions of 1:2, 1:4, 1:8 and 1:16 of this filtrate were tested, corresponding to nominal loading rates of 100, 50, 25, 12.5 and 6.25 mg test item/L (spacing factor 2.0) corresponding to the arithmetic mean measured concentrations of 25.9, 13.1, 6.70, 3.29 and 1.64 mg test item/L.

The quantification of the test item bis[4-(tert-butyl)benzoato-O]hydroxyaluminium in the test samples was performed via measurement of Total Organic Carbon (TOC).

The concentrations of carbon were determined in the duplicate test media samples from all test concentrations (test item stock solution and its dilutions) and the duplicate control samples from all sampling times. The measured carbon concentrations in the two lowest test concentrations were found to be below the limit of quantification therefore the measured test item concentrations were extrapolated using the mean recovery rates of the three highest test concentrations for these two treatment groups.

The measured TOC values were compared with the nominal TOC values of each test concentration calculated using the test item carbon content of 65.75%, to recalculate the mean measured concentrations of the test item in the test samples.

The mean measured values are

25.9 mg Test Item/L in the filtrate

13.1 mg Test Item /L in the 1:2 dilution of filtrate

6.70 mg Test Item /L in the 1:4 dilution of filtrate

3.29 mg Test Item /L in the 1:8 dilution of filtrate and

1.64 mg Test Item /L in the 1:16 dilution of filtrate.

The 72-hour EyL50 was calculated to be 70.7 mg test item/L and the ErL50 > 100 mg test item/L, corresponding to mean measured concentrations of 18.4 and > 25.9 mg test item/L, respectively. The 72-hour EyL10 was calculated to be 52.6 mg test item/L and the ErL10 67.7 mg test item/L, corresponding to mean measured concentrations of 13.8 and 17.6 mg test item/L, respectively. The 72-hour NOEyL was determined to be 50 mg test item/L and the associated 72-hour LOEyL was 100 mg test item/L, corresponding to mean measured concentrations of 13.1 and 25.9 mg test item/L, respectively. The 72-hour NOErL was determined to be 50 mg test item/L and the associated 72-hour LOErL was 100 mg test item/L, corresponding to mean measured concentrations of 13.1 and 25.9 mg test item/L, respectively.

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a current reference study to assure that the test conditions are reliable.

No observations were made which might cause doubts concerning the validity of the study outcome. All validity criteria were met.The result of the test is considered valid.