Reaction mass of [2,2,4-trimethyl-1-(2-methylpropanoyloxy)pentan-3-yl] benzoate and [2,2,4-trimethyl-3-(2-methylpropanoyloxy)pentyl] 2-methylpropanoate and (3-benzoyloxy-2,2,4-trimethylpentyl) benzoate

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18.03.2016-08.05.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: ASTM E 1193-97

GLP compliance:

yes

Specific details on test material used for the study:

Physical state/Appearance: Clear colorless liquid
Batch: V046414101
Purity: 100%
Expiry Date: 20 November 2016
Storage Conditions: Room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

Water samples were taken from the control and each surviving test group (replicates pooled) for quantitative analysis. Samples of the fresh test preparations were taken on Days 0, 5, 12 and 20 and of the expired test preparations on Days 1, 6, 13 and 21. Samples were either analysed on the day of sampling or stored at approximately 4 °C prior to analysis. Duplicate samples were taken and stored frozen for further analysis if necessary.
The method of analysis, recovery and test preparation analyses are described in Appendix 2 of the report.

Vehicle:

not specified

Details on test solutions:

Reconstituted Water – Elendt M7 Medium
Ingredient Final Concentration (mg/L)
H3BO3 0.715
MnCl2.4H2O 0.090
LiCl 0.077
RbCl 0.018
SrCl2.6H2O 0.038
NaBr 0.004
Na2MoO4.2H2O 0.016
CuCl2.2H2O 0.004
ZnCl2 0.013
CoCl2.6H2O 0.010
KI 0.0033
Na2SeO3 0.0022
NH4VO3 0.00058
Na2EDTA.2H2O 0.625
FeSO4.7H2O 0.249
CaCl2.2H2O 293.8
NaHCO3 64.8
MgSO4.7H2O 123.3
Na2SiO3.9H2O 10
KCl 5.8
NaNO3 0.274
K2HPO4 0.184
KH2PO4 0.143
Thiamine hydrochloride 0.075
Cyanocobalamine (vitamin B12) 0.0010
D(+) biotin (vitamin H) 0.00075

The pH of the prepared media was 7.9 ± 0.3 and stored at approximately 21 ºC.

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium (see Appendix 1) in a temperature controlled room at approximately 20 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing by randomly assigning to the test vessels. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Test type:

not specified

Water media type:

other: reconstituted water (Elendt M7 medium)

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

At the end of the test, the length of each surviving parent animal was determined.

Hardness:

not specified

Test temperature:

18 to 22 °C

pH:

7.6-8.2

Dissolved oxygen:

All >1.5 O2/L

Salinity:

not specified

Conductivity:

not specified

Nominal and measured concentrations:

nominal concentrations of 1.0, 3.2, 10, 32 and 100% v/v

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.62 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

0.56 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.47 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.52 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.15 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.15 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.15 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth

Key result

Duration:

21 d

Dose descriptor:

other: Chronic value (ChV)

Effect conc.:

0.28 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

reproduction

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Daphnia magna to the test item resulted in significant mortalities at the test concentration of 1.5 mg/L resulting in 100% mortalities by Day 5.
The 21-Day EC50 (immobilization) value, based geometric mean measured concentrations, for the parental daphnia generation (P1) was calculated to be 1.0 mg/L. Due to the nature of the data, no confidence limits could be determined.
The 21-Day EC50 (reproduction) based on geometric mean measured concentrations was 0.56 mg/L. Due to the nature of the data, no confidence limits could be determined.
The "Lowest Observed Effect Concentration" (LOEC) and the "No Observed Effect Concentration" (NOEC) based on geometric mean measured concentrations were 0.52 and 0.15 mg/L respectively.
The Chronic Value (ChV) was determined to be 0.28 mg/L.

Executive summary:

1.1 Introduction

A study was performed to assess the chronic toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the ASTM E 1193-97 Guideline.

1.2 Methods

Based on the results of preliminary work performed in a previous study (Envigo Study Number 41500225), the test media was prepared using a saturated solution method of preparation.

Based on the results of a previous test (Harlan Study Number 41202706), Daphnia magna were exposed (10 replicates of a single daphnid per group) to solutions of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution for a period of 21 days. The test solutions were renewed daily throughout the test. The test item solutions were prepared by stirring an excess (100 mg/L) of test item in test media using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by centrifugation at 10000 g for 30 minutes to produce a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups.

The numbers of live and dead adult daphnia and young daphnids (live and dead) were determined daily. The daphnia were fed daily with a mixture of algal suspension and Tetramin® flake food suspension.

1.3 Results

Analysis of the fresh test preparations on Days 0, 5, 12 and 20 showed measured test concentrations to range from 0.025 to 2.3 mg/L. A decline in measured test concentration of the aged test preparations on Days 1, 6, 13 and 21 was observed to between less than the limit of quantification (LOQ) of the analytical method, determined to be 0.018 mg/L, and 1.3 mg/L. It was considered appropriate to calculate the results based on the geometric mean measured test concentration only in order to give a “worst case” analysis of the data.

Exposure of Daphnia magna to the test item gave the following results based on the geometric mean measured test concentrations:

Endpoint		Concentration (mg/L)
Immobilization	EC10	0.62
	95% confidence limits	Not determined
	EC50	1.0
	95% confidence limits	Not determined
Reproduction	EC10	0.47
	95% confidence limits	Not determined
	EC50	0.56
	95% confidence limits	Not determined
No observed effect concentration (NOEC)		0.15
Lowest observed effect concetration (LOEC)		0.52
Chronic value (ChV)		0.28

Description of key information

Exposure of Daphnia magna to the test item resulted in significant mortalities at the test concentration of 1.5 mg/L resulting in 100% mortalities by Day 5.

The 21-Day EC50 (immobilization) value, based geometric mean measured concentrations, for the parental daphnia generation (P1) was calculated to be 1.0 mg/L. Due to the nature of the data, no confidence limits could be determined.

The 21-Day EC50 (reproduction) based on geometric mean measured concentrations was 0.56 mg/L. Due to the nature of the data, no confidence limits could be determined.

The "Lowest Observed Effect Concentration" (LOEC) and the "No Observed Effect Concentration" (NOEC) based on geometric mean measured concentrations were 0.52 and 0.15 mg/L respectively.

The Chronic Value (ChV) was determined to be 0.28 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.15 mg/L

Marine water invertebrates

Marine water invertebrates

Effect concentration:

0.15 mg/L