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

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:

2018-07-09 - 2018-07-26 (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 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

OECD Guideline for Testing of Chemicals No. 202: "Daphnia sp., Acute Immobilisation Test" adopted April 13, 2004

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

Commission Regulation (EC) No 440/2008, Annex, Part C, C.2.: "Daphnia sp. Acute Immobilisation Test", Official Journal of the European Union (EN), dated May 30, 2008

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, 50, 25, 12.5 and 6.25 mg test item/L (spacing factor 2) and control
- Sampling method: The samples were taken from the biological phase of the study. Duplicate samples from the freshly prepared test media of all test concentrations and 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 were collected at the end of the test (after 48 hours) 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 is not well soluble in test water. To avoid physical effects of undissolved test item on daphnids no concentrations above the solubility limit of the test item in test water were tested.
Therefore, a supersaturated stock suspension of the test item was prepared by suspending test item in test water at a concentration of 100 mg test item/L. The stock solution suspension was shaken overhead for approximately 48 hours 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). Adequate volumes of this filtrate were mixed into test water to obtain the desired dilutions of 1:2, 1:4, 1:8 and 1:16.
The test media were prepared just before introduction of the daphnids (= 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):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (Straus)
- Strain/clone: clone 5
- Sex: Female
- Source: The daphnids introduced in the test were taken from ibacon's in house laboratory culture.
- Feeding during test: no
- Food type: Green algae (Desmodesmus subspicatus) freshly grown in the laboratories of ibacon.
- Amount: not specified
- Frequency: The daphnids in the stock culture were fed at least on all working days.

ACCLIMATION
- Acclimation period: Was not necessary, since the test was performed in the same medium as the culturing.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
The daphnids introduced in the test were 1.75 to 20 hours old. The method for preparation and collection of early instars was not specified.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Test temperature:

20.5 to 20.7 °C at test start;
21.2 °C at test end

pH:

7.9 at test start;
7.8 to 7.9 at test end

Dissolved oxygen:

8.1 to 8.9 mg/L at test start;
8.7 mg/L 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): 29.9, 14.5, 7.29, 3.67 and 1.84 mg test item/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Beakers
- Type (delete if not applicable): Open. The test vessels were only covered with a lid to reduce the loss of water due to evaporation and to avoid the entry of dust into the solutions.
- Material, size, headspace, fill volume: Glass beakers of 100 mL volume containing approximately 60 mL of test medium.
- Aeration: No.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Water (Elendt "M4"): Analytical grade salts and additives were added in deionised water at defined nominal concentrations (see "Any other information on materials and methods incl. tables").
- Alkalinity: 0.9 mmol/L
- Conductivity: < 5 µScm-1
- Culture medium different from test medium: No. The cultivation of the parental daphnids was performed in Elendt M4 medium also.

OTHER TEST CONDITIONS
- Adjustment of pH: No.
- Photoperiod: 16 h light : 8 h dark
- Light intensity: The light intensity was 160 to 450 lux (measured once during the test).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The mobility of the daphnids was determined by visual observation after 24 and 48 hours. Those animals that are not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile (even if they could still move their antennae).

VEHICLE CONTROL PERFORMED: No

RANGE-FINDING STUDY
- 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, corresponding to nominal loading rates of 100, 10, 1.0 and 0.10 mg test item/L.
- Results used to determine the conditions for the definitive study: In the range-finding study all animals were immobile at nominal 100 mg/L but no immobilisation was observed at the lower concentrations of nominal 10, 1.0 and 0.1 mg/L.

Reference substance (positive control):

yes

Remarks:

For the evaluation of the quality of the Daphnia clone and the consistency of the experimental conditions, the reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

57.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% conf. interval: not determinable

Effect concentrations 2

Duration:

48 h

Dose descriptor:

other: EL20

Effect conc.:

53.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% conf. interval: not determinable

Effect concentrations 3

Duration:

48 h

Dose descriptor:

EL10

Effect conc.:

51.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% conf. interval: not determinable

Effect concentrations 4

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 5

Duration:

48 h

Dose descriptor:

LOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 6

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

16.79 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% conf. interval: not determinable

Effect concentrations 7

Duration:

48 h

Dose descriptor:

other: EC20

Effect conc.:

15.67 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% conf. interval: not determinable

Effect concentrations 8

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

15.05 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% conf. interval: not determinable

Effect concentrations 9

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

14.5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 10

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

29.9 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 11

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

95.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% conf. interval: 90.1 - > 100 mg/L

Effect concentrations 12

Duration:

24 h

Dose descriptor:

other: EL20

Effect conc.:

87.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% conf. interval: 83.1 - 92.6 mg/L

Effect concentrations 13

Duration:

24 h

Dose descriptor:

EL10

Effect conc.:

83.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% conf. interval: 79.3 - 88.2 mg/L

Effect concentrations 14

Duration:

24 h

Dose descriptor:

NOELR

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 15

Duration:

24 h

Dose descriptor:

LOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 16

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

28.44 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% conf. interval: 26.93 - 30.10 mg/L

Effect concentrations 17

Duration:

24 h

Dose descriptor:

other: EC20

Effect conc.:

26.19 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% conf. interval: 24.85 - 27.68 mg/L

Effect concentrations 18

Duration:

24 h

Dose descriptor:

EC10

Effect conc.:

24.97 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% conf. interval: 23.70 - 26.35 mg/L

Effect concentrations 19

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

14.5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 20

Duration:

24 h

Dose descriptor:

LOEC

Effect conc.:

29.9 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: No.
- Mortality of control: Was 0 % and furthermore no daphnid showed signs of disease or stress.
- Other adverse effects control: No (see above).
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: There were no remarkable observations. All test media were clear and colourless.
- Effect concentrations exceeding solubility of substance in test medium: No.

Results with reference substance (positive control):

- Results with reference substance valid? Yes.
- Limit test: No.
- Dose-response test: Yes.
- Other: In the most recent test with the reference item potassium dichromate performed in January 2018 (study code 88299220), the EC50 after 24 hours was determined to be 1.06 mg test item/L, indicating that the sensitivity of the daphnids was consistent with the level proposed by the OECD 202 guideline (EC50-24 h between 0.6 and 2.1 mg potassium dichromate/L). The EC50 after 48 hours was determined to be 1.02 mg potassium dichromate/L.
In the reference test performed in June 2017 (study code 88298220) with the reference item potassium dichromate, the EC50 after 24 hours was determined to be 1.58 mg test item/L and the EC50 after 48 hours was determined to be 1.07 mg test item/L.

Reported statistics and error estimates:

The 24-hour and 48-hour EC50, EC20 and EC10 and the 95 % confidence limits were calculated by logit analysis.
The NOEL and LOEL after 24 and 48 hours were calculated by Step-down Cochran Armitage test procedure.
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 202 and EU Method C.2 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 daphnids.
The toxic effect of the test item bis[4-(tert-butyl)benzoato-O]hydroxyaluminium to Daphnia magna was assessed in a static concentration-response test. The 48-hour NOEL was determined to be 50 mg test item/L, corresponding to a mean measured concentration of 14.5 mg test item/L. The 48-hour LOEL was determined to be 100 mg test item/L and the 48-hour EL50 value was calculated to be 57.5 mg test item/L, corresponding to mean measured concentrations of 14.5 and 16.8 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 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 48 -hour EL50 of 57.5 mg test item/L, corresponding to an 48 -hour EC50 of 16.8 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 study was to determine the influence of the test item bis[4-(tert-butyl)benzoato-O]hydroxyaluminium on the mobility of Daphnia magna. For this purpose, young daphnids (< 24 hours old) were exposed in a static test to various concentrations under defined conditions for 48 hours. The study encompassed 6 treatment groups (5 dose rates of the test item and a control) each containing 20 individuals. The mobility of the daphnids was determined by visual observation after 24 and 48 hours.

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 and mean measured concentrations of 29.9, 14.5, 7.29, 3.67 and 1.84 mg test item/L

After 48 hours of exposure no immobilisation of the test animals was observed in the control and up to and including the test item loading rate of 25 mg test item/L. At the loading rate of 50 mg test item/L one animal was immobile. At the loading rate of 100 mg test item/L, all animals were immobile.

The quantification of the test item 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

29.9 mg Test Item/L in the filtrate

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

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

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

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

The 48-hour NOEL was determined to be 50 mg test item/L, corresponding to a mean measured concentration of 14.5 mg test item/L. The 48-hour LOEL was determined to be 100 mg test item/L and the 48-hour EL50 value was calculated to be 57.5 mg test item/L, corresponding to mean measured concentrations of 14.5 and 16.8 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 mean measured test item concentrations were used for the result evaluation.

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.