Vinyl 4-(1,1-dimethylethyl)benzoate

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:

27 Okt - 29 Okt 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

adopted April 13, 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Vinyl 4-(1,1-dimethylethyl)benzoate

EC Number:

239-510-5

EC Name:

Vinyl 4-(1,1-dimethylethyl)benzoate

Cas Number:

15484-80-7

Molecular formula:

C13H16O2

IUPAC Name:

ethenyl 4-tert-butylbenzoate

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method: One sample per freshly prepared stock solution in DMF was taken at the start of the test. Triplicate samples from the freshly prepared test media of all test concentrations and the controls were taken at the start of the test and at day 1.
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, triplicate samples from the aged test media of all test concentrations and the controls were collected at day 1 (after 24 hours of exposure) and at the end of the test by pouring together the contents of the test beakers of each treatment.

- Sample storage conditions before analysis: deep frozen(≤ - 20 °C), protected from light

Test solutions

Vehicle:

yes

Remarks:

Reconstituted water with the solvent dimethylformamide (DMF) at a concentration of 100 µL/L.

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
A stock solution of 700 mg test item/L was prepared by dissolving 14.2 and 14.1 mg of the test item in 20.3 and 20.1 mL dimethylformamide. This stock solution was diluted with the solvent using the same spacing factor as used to define the nominal test concentrations to allow the addition of the same volume of solvent (100 µL/L) to each test solution. The solvent additive was chosen based upon its solubility properties and its relative lack of toxicity to daphnids. A defined volume of the stock solution and its dilutions was mixed into test water to obtain the test media of the desired test concentrations.
The test media were prepared just before introduction of the daphnids (= start of the test) and test medium renewal on Day 1.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
Daphnia magna (Straus), clone 5
Age at Test Start: From 2.5 to 22.5 hours old
Source: The daphnids introduced in the test were taken from ibacon's in-house laboratory culture.
The daphnids were bred in the laboratories of ibacon under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in Elendt M4 medium. The test organisms were not first brood progeny. The daphnids in the stock culture were fed at least on all working days with green algae (Desmodesmus subspicatus) freshly grown in the laboratories of ibacon.
Feeding during the test: none
Acclimatisation: was not necessary, since the test was performed in the same medium as the culturing.

Study design

Test type:

semi-static

Water media type:

other: Reconstituted water, Elendt "M4"

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

Water Hardness: 2.5 mmol/L (= 250 mg/L) as CaCO3

Test temperature:

Water Temperature: 19.8 to 20.2 °C in the freshly prepared media;
19.3 to 20.4°C in the aged test media

pH:

8.1 in the freshly prepared media;
8.0 to 8.1 in the aged test media;
and thus the pH-value did not vary by more than 1.5 units

Dissolved oxygen:

8.9 to 9.1 mg/L in the freshly prepared media;

Nominal and measured concentrations:

nominal: 0.07, 0.032, 0.014, 0.007 and 0.003 mg test item/L (spacing factor 2.2) (each in 100 µL DMF/L), a solvent control (100 µL DMF/L) corresponding to following time weighted average measured concentrations of the test item:
0.058, 0.027, 0.012, 0.006 and 0.003 mg test item/L (each in 100 µL DMF/L), a solvent control (100 µL DMF/L), and a control.

The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part.

Details on test conditions:

Test Vessel:Glass beakers of approximately 110 mL volume containing as much test medium as possible (i.e. the remaining head space was reduced to a technical possible minimum of some millilitres), kept closed during the whole period of the study with a conical glass stopper to avoid loss of the test item due to volatilisation.
Light Regime: 16 h light : 8 h dark
Light Intensity: The light intensity was 720 to 740 lux (measured once during the test).
Introduction of Daphnids: 20 daphnids per control and test concentration, divided into 4 groups of 5 animals, each group in one test vessel. Daphnia were assigned to the test vessels randomly.
Replicates: The test was performed with four replicates per treatment group.. Exposure Time: 48 hours
Test Procedure: A semi-static test system with a test medium renewal on Day 1 was chosen to keep the concentrations of test item as constant as possible in the test media.
Feeding: None
Sampling: The concentrations of the test item were analysed in two of the triplicate test media samples from all test concentrations and in one of the triplicate control and solvent control samples from all sampling times.

Test Parameter:
Immobility: 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).

Pre-experiments were performed to determine a suitable concentration range and to establish suitable methods to prepare the test solutions.

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

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.028 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

0.018 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.012 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

0.027 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Signs of Intoxication after 48 Hours:
After 48 hours of exposure no immobilisation was observed in the control, the solvent control and up to the concentration of 0.0119 mg test item/L. At the concentration of 0.0268 mg test item/L, 9 daphnids were immobile and all 20 daphnids were immobile at the highest concentration of 0.0577 mg test item/L

Results with reference substance (positive control):

Results of the most recent Test with the Reference Item Potassium Dichromate
Dose response test: 0.40, 0.60, 0.89, 1.33 and 2 mg/L
EC50 24h: 1.21 mg/L
NOEC 24 h: 0.60 mg/L
LOEC 24 h: 0.89 mg/L
EC50 48 h: 0.844 mg/L
NOEC 48 h: 040 mg/L
LOEC 48 h: 0.60 mg/L

The reference test was valid:The results indicate 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).

Reported statistics and error estimates:

The 24-hour and 48-hour EC50, EC20 and EC10 and the 95 % confidence limits were calculated using Weibull analysis.
The NOEC and LOEC after 24 and 48 hours were determined using Fisher`s Exact Binomial Test with Bonferroni Correction (24) and Step-down Cochran Armitage test procedure (48).
The software used to perform the statistical analysis was ToxRat Professional, Version 3.3.0, ToxRat Solutions GmbH.

Any other information on results incl. tables

Validity criteria

Criterion	value required	value obtained	fullfilled
Control immobilisation rate	< 10%	0%	yes
Dissolved oxygen concentration	at the end of the test >= 3 mg O2/L	≥ 8.8 mg O2/L in in all treatment groups at the end of the test	yes

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

see any other information on results

Conclusions:

The toxic effect of the test item to Daphnia magna was assessed in a semi-static concentration-response test. The 48-hour NOEC was determined to be 0.0119 mg test item/L. The 48-hour LOEC was determined to be 0.0268 mg test item/L and the 48-hour EC50 value was calculated to be 0.02811 mg test item/L.
The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to time weighted average concentrations, since the test item concentrations were not within ± 20 % of the nominal or measured initial concentrations during the test.

Executive summary:

Test Species: Daphnia magna, clone 5; 2.5 to 22.5 hours old

Test Design: This study encompassed 7 treatment groups (5 dose rates of the test item, a solvent control and a control) each containing 20 individuals. The mobility of the daphnids was determined in a semi-static 48-hour test by visual observation after 24 and 48 hours.

Endpoints: Number of immobile organisms after 24 and 48 hours

Test Concentrations:Nominal 0.07, 0.032, 0.014, 0.007 and 0.003 mg test item/L (spacing factor 2.2) (each in 100 µL DMF/L), a solvent control (100 µL DMF/L) corresponding to following time weighted average measured concentrations of the test item:0.058, 0.027, 0.012, 0.006 and 0.003 mg test item/L (each in 100 µL DMF/L), a solvent control (100 µL DMF/L), and a control.

Test Conditions: Water temperature: 19.3 to 20.4 °C; pH value: 8.0 to 8.1; dissolved oxygen concentration: 8.8 to 9.1 mg/L; photoperiod: 16 h light - 8 h dark; light intensity: 720 to 740 lux; and thus were within the ranges requested by guideline OECD 202.

Results:

After 48 hours of exposure no immobilisation was observed in the control, the solvent control and up to the concentration of 0.0119 mg test item/L. At the concentration of 0.0268 mg test item/L, 9 daphnids were immobile and all 20 daphnids were immobile at the highest concentration of 0.0577 mg test item/L.

Parameter
	after 24 h	after 48 h
EC50 mg test item/L	not determinable	0.02811
EC20 mg test item/L	not determinable	0.02166
EC10 mg test item/L	0.03427	0.01823
NOEC mg test item/L	≥ 0.0577	0.0119
LOEC mg test item/L	> 0.0577	0.0268

Analytical Results:

The quantification of the test item in the test samples was performed using gas chromatography with MS/MS detection.

In the freshly prepared test media at the test start and at the renewal of the test media 99 % of the nominal test concentrations were found (average of all test concentrations). In the aged test media after 24 hours test duration on day 1 and at test end, 72 % of the nominal value was determined (average of all test concentrations).

Conclusion:The toxic effect of the test item to Daphnia magna was assessed in a semi-static concentration-response test. The 48-hour NOEC was determined to be 0.0119 mg test item/L. The 48-hour LOEC was determined to be 0.0268 mg test item/L and the 48-hour EC₅₀ value was calculated to be 0.02811 mg test item/L.

The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to time weighted average concentrations, since the test item concentrations were not within ± 20 % of the nominal or measured initial concentrations during the test.