4,4'-isopropylidenedicyclohexanol

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:

24 March 2016 to 09 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 7095
- Expiration date of the lot/batch: 31 December 2016
- Purity: 95.6%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: verified by chemical analysis on Days 0, 7, 14 and 20 (fresh media) and on Days 1, 8, 15 and 21 (old media)

Analytical monitoring:

yes

Remarks:

gas chromatography with mass spectrometry

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
A nominal amount of test item (1100 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Gelman Acrocap filter (first approximate 500 mL discarded in order to pre-condition the filter) to give a 100% v/v saturated solution stock solution. A series of dilutions was made from this 100% v/v saturated solution to give the test concentrations of 3.13, 6.25, 12.5, 25 and 50% v/v saturated solution.
Each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Source: in-house laboratory cultures
- Age: less than 24 hours old
- Feeding during test
- Food type: algal suspension (Desmodesmus subspicatus) and flake food
- Amount: 5 to 15 μL of an algal suspension and 20 μL of flake food
- Frequency: daily

Test type:

semi-static

Water media type:

other: Reconstituted Water

Limit test:

no

Total exposure duration:

21 d

Hardness:

232 to 272 mg/L as CaCO3

Test temperature:

approximately 21 °C

pH:

7.7 - 8.1

Dissolved oxygen:

≥8.1 mg O2/L

Nominal and measured concentrations:

Initial experiment: 1.25, 2.5, 5.0, 10 and 20 mg/L.
Definitive Test: 3.13, 6.25, 12.5, 25 and 50% v/v saturated solution

Details on test conditions:

TEST SYSTEM
- Test vessel: glass vessels
- Type (delete if not applicable): covered with a plastic lid
- Material, size, headspace, fill volume: 100 mL of the test preparation in 150 mL glass vessels.
- Aeration: The diluent water was aerated prior to use, however, the test vessels were not aerated.
- Renewal rate of test solution: daily.
- No. of organisms per vessel: 1
- No. of vessels per concentration: 10
- No. of vessels per control: 10

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods
- Light intensity: 480 to 586 lux
- Food: 5 to 15 μL of an algal suspension (Desmodesmus subspicatus) and 20 μL of Tetramin® flake food suspension daily, approximately 0.1 to 0.2 mg carbon/daphnid/day

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: 3.13, 6.25, 12.5, 25 and 50% v/v saturated solution.

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 30 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 30 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 30 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 30 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

30 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Details on results:

- Lethal Effects on the Parental Generation (P1): Mortalities were observed in each test concentration. However, statistical analysis of the mortality data using the Fisher’s Exact test showed that the observed mortalities were not significantly different (P≥0.05) when compared to the control group. As a result, for the purposes of the results, all adult mortalities were classed as inadvertent/accidental, not as a result of the test item.

- Sub-lethal Effects on the Parental Generation (P1): After 21 days the length of each surviving adult was determined. The results showed that there were no statistically significant differences (P≥ 0.05) between the control and each test group in terms of length of the daphnids after 21 days exposure to the test item.

- Effects on Reproduction: Analysis of the data obtained on Day 21 showed that the numbers of live young produced per adult by the control group were not significantly different (P≥0.05) from the control and each test group.

- Effects on the Filial Generation (F1): Information on the effects of the test item on the F1 generation is limited, since, by study design, the young are removed soon after liberation from the brood pouch. However, an assessment made at each media renewal showed the "filial" daphnids produced by all the 3.13, 6.25 and 12.5% v/v saturated solution test groups were in the same general condition as the young produced by the controls over the duration of the test. However, the young produced by the 25 and 50% v/v saturated solution test groups were observed to be small/pale when compared to the control young on Days 18, 19 and 21. Aborted eggs were also observed in the 50% v/v saturated solution test group on Day 19.
Small young were observed in the control group on Day 21.
Young were first produced in the control group on Day 7 of the test.
There were no dead young observed in all control and treatment groups surviving to maturation.

- No Observed Effect Concentration: NOEC was 30 mg/L as there were no significant mortalities (immobilization) observed in the parental generation (P1) and there were no significant differences (P≥0.05) in terms of the number of live young produced per adult when compared to the control after 21 days.

- Verification of Test Concentrations: Analysis of the fresh test preparations on Days 0, 7, 14 and 20 showed measured test concentrations to range from 1.35 to 32 mg/L. There was no significant change in the measured concentrations of the aged test preparations on Days 1, 8, 15 and 21 and so the results are based on the average measured concentrations of the fresh test preparation only.

- Observations on Test Item Solubility: At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

Reported statistics and error estimates:

- 21-Day EC50 value was given by inspection of the immobilization data
- EC50 (reproduction) value after 21 days was estimated by inspection of the data
- NOEC the numbers of live young produced per adult over the duration of the test for the control and each test group were compared using the Williams Multiple Sequential t-test Procedure.
- Daphnia length data, determined for the surviving daphnids on termination of the test, were compared using one way analysis of variance incorporating Bartlett’s test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett’s multiple comparison procedure for comparing several treatments with a control (Dunnett 1955).
- Statistical analyses was performed using the SAS computer software package (SAS 1999 - 2001) or the ToxRat Professional computer software package (TOXRAT).

Validation Criteria

The following validation criteria were achieved during the test:

Required       Actual

a) Control mortality                                 ≤ 20%                   20%

b) Mean number of live young per       ≥ 60 after 21 days       135

surviving adult (control group)

c) Coefficient of variation for control       ≤ 25%                      6%

group*

d) Ephippia produced in the controls        0                                0

e) Dissolved oxygen                                > 3 mg O2/L             ≥8.1 mg O2/L

f) pH (control group)                                6 to 9                      7.4 to 8.4

Variation                                     ≤1.5                       1.0

∗ Based on total number of living offspring per parent animal alive at the end of the test

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Daphnia magna to the test item resulted in no significant mortalities at all the concentrations employed during the test.
The 21-Day EC50 (immobilization) value, based on the average measured concentration of the freshly prepared test preparations, for the parental daphnia generation (P1) was estimated to be greater than 30 mg/L.
No significant impairment of reproduction was observed at the concentrations employed during the test.
The 21-Day EC50 (reproduction) based on the average measured concentration of the freshly prepared test preparations was greater than 30 mg/L.
The "No Observed Effect Concentration" (NOEC) based on the average measured concentration of the freshly prepared test preparations was 30 mg/L.

Description of key information

21-Day Daphnia magna Reproduction Test

EC₅₀ (immobilization) (P1) > 30 mg/L.

EC₅₀ (reproduction) > 30 mg/L.

NOEC: 30 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

30 mg/L

Additional information

A determination of the chronic toxicity to aquatic invertebrates of HBPA was conducted (Envigo Research Limited, 2016, Study MC06VS). The study was performed in accordance with EU Method C.20 and OECD test guideline 211, and in compliance with GLP.

Based on the results of preliminary work, Daphnia magna (10 replicates of a single daphnid per group) were exposed to a solution of HBPA at nominal concentrations of 3.13, 6.25, 12.5, 25 and 50% v/v saturated solution for a period of 21 days. The test solutions were renewed daily throughout the test.

Analysis of the fresh test preparations showed measured test concentrations to range from 1.35 to 32 mg/L. There was no significant change in the measured concentrations of the aged test preparations and so the results are based on the average measured concentrations of the fresh test preparation only.

Exposure of Daphnia magna to HBPA resulted in no significant mortalities at all the concentrations employed during the test. The 21-Day immobilization and reproduction EC50 were to be estimated to be greater than 30 mg/L, whereas the 21-Day NOEC was 30 mg/Lon the basis that at this test concentration there were no significant mortalities (immobilization) observed in the parental generation (P1) and that there were no significant differences (P≥0.05) between the control and the 30 mg/L test group in terms of numbers of live young produced per adult by Day 21.