Ethyl trans-2,2,6-trimethylcyclohexanecarboxylate

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:

12 February 1996 to 4 March 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals (1 984) No. 202 "Daphnia sp., Acute lmmobilisation Test and Reproduction Test".

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Water samples were taken from the solvent control and each surviving test group (replicates pooled) for quantitative analysis. Samples of freshly prepared solutions were taken on day 0 and of the expired test solutions on days 2, 4, 7, 9, 11, 14, 16, 18 and 21. Chemical analysis was also carried out on the solvent stock solutions used to prepare the test series on days 0, 2, 4, 7, 9, 11 and 14.

Vehicle:

yes

Details on test solutions:

For the purpose of the definitive study the test material was prepared using a preliminary solution in ethanol.
Amounts of test material (250 and 800 mg) were each separately dissolved in solvent and the volume adjusted to 25 ml to give solvent stock solutions of 250 and 800 mg/25 ml. Serial dilutions were made to give further solvent stock solutions of 8.0, 25 and 80 mg/25 ml. An aliquot (500 ul) of each solvent stock solution was separately dispersed in 5 litres (final volume) of reconstituted water to give the test series of 0.032, 0.10, 0.32, 1.0 and 3.2 mg/l.

Test organisms (species):

Daphnia magna

Details on test organisms:

Test Species
The test was carried out using 1st instar Daphnia magna. Daphnia magna were maintained in a laboratory culture originating from a strain supplied by the lnstitut National de Recherche Chimique Appliquée (IRCHA) France.
Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water at a temperature of 21°C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle. Each culture was fed daily with a suspension of mixed algae (predominantly Chlorella spp.). Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated 24 hours prior to the initiation of the test, the young daphnids produced overnight were then removed for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Test temperature:

21.0°C

pH:

7.6-7.8

Dissolved oxygen:

7.8-8.4 mg/ L

Salinity:

Not applicable

Nominal and measured concentrations:

Based on the results of an acute toxicity test with Daphnia magna (Project Number 249/031) the following nominal test concentrations were assigned to the study : 0.032, 0.10, 0.32, 1.0 and 3.2 mg/l.
Chemical analysis of the solvent stock solutions up to, and including day 14 showed the measured test concentrations to be near nominal with the exception of the 8.0 mg/25 ml solvent stock solution on days 0 and 7 which gave measured test concentrations of 127 and 121% of nominal respectively. These values are considered to be due to sampling and/or analytical variation. Analysis of the solvent stock solutions was not performed after day 14 as it was considered that sufficient data had been obtained to show that preparation of the solvent stock solutions and the fresh media prepared from them was satisfactory.Analysis of the test solutions at 0 hours showed the measured test concentrations to be near nominal with the exception of the 0.10 mg/l test concentration which showed a measured test concentration of 123% of nominal. This value is considered to be due to sampling and/or analytical variation. The spent test media showed a decline in measured test concentrations over the study duration with values ranging between 68 - 99% of nominal.

Details on test conditions:

For each concentration 10 daphnids were placed in 500 ml of the prepared test solutions in 500 ml glass conical flasks which were then sealed with ground glass stoppers to reduce evaporation and possible losses of test material due to its volatile nature. For each test and control group four replicate test vessels were prepared.
The control and solvent control vessels were spatially separated from the test vessels in order to prevent any possible contamination due to the volatile nature of the test material. The flasks were maintained at 21°C with a photoperiod of 16 hours light and 8 hours darkness for a period of 21 days. The daphnids were not individually identified and the test vessels were not aerated. The diluent water only was aerated prior to use.The control and the solvent control groups were maintained under identical conditions but not exposed to the test material. The solvent control group was exposed to 100 ul/ l of ethanol.

The test solutions were renewed 3 times per week on days 0, 2, 4, 7, 9, 1 I , 14, 16 and 18. The adult Daphnia were transferred to fresh media by wide-bore pipette before the contents of each vessel were passed through a fine mesh. Young daphnids (live and dead) and any unhatched eggs were collected on the mesh and counted using a stereo microscope before being discarded.

Each daphnid received approximately 5 - 10 ul of a mixed unicellular algal culture (equivalent to approximately 2.92 x 10^9 cells/ml), daily. Feeding was at a level to maintain a green tinge in the test solutions thereby ensuring that food was available continuously. Equal amounts of food were given to each daphnid. The numbers of live and dead Daphnia of the "Parental" (P1) generation were counted daily. An assessment of the general condition and size of the parental Daphnia as compared with the controls and the numbers of Daphnia with eggs or young in the brood pouch was determined at each test media renewal together with the numbers of live and dead "filial" (F1) Daphnia. The general condition of the "filial" Daphnia was assessed as compared to the controls and the number of discarded unhatched eggs was also determined at each test media renewal.Young daphnids were considered to be dead if no sign of movement was apparent during microscopic examination. Adult Daphnia which were unable to swim for approximately 15 seconds after gentle agitation (ie. immobile), were considered to be dead. An immobilisation criterion for the young daphnids was considered to be inappropriate due to the large numbers of off-spring produced in the flasks.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

Lethal Effects on the Parental Generation (P1)
Mortality (immobilisation) occurred within the first 11 days of exposure at the highest test concentration of 3.2 mg/l. No mortalities occurred at the test concentrations of 0.032, 0.10, 0.32 and 1.0 mg/l throughout the duration of the study. The EC50 (immobilisation) values based on nominal test concentrations were calculated by the moving average method of Thompson (1947): see section "any other information on results incl. tables" for details. Although it was not possible to calculate the 24 and 48-Hour EC50, values (since less than 50% mortalities were observed) the observed mortalities were consistent with the results of the acute toxicity to 1 st instar Daphnia magna (Project No 249/031) where the 48-Hour EC, was calculated to be 3.2 mg/l.
There was a significant effect on size and colouration of the daphnids as a result of exposure to ET-344-SP in that the surviving daphnids at the test concentration of 3.2 mg/l were markedly paler in colour than the control animals on day 4 and smaller in size and paler in colour on days 7 and 9 until 100% immobilisation occurred by day 11.The daphnids at the test concentrations of 0.032, 0.1 0, 0.32 and 1.0 mg/l were observed to be the same size and colour as the control animals.
Sub-lethal Effects on the Parental Generation (P1)
After both 14 and 21 days there were no statistically significant differences between the solvent control and the 0.032, 0.10, 0.32 and 1.0 mg/l test groups in terms of the number of young produced per adult.The EC50 (reproduction) after 14 and 21 days can only be estimated, given the unsuitable nature of the data for standard calculation of EC50 values. The estimated EC50 (reproduction) for 14 and 21 days exposure is considered to be between 1.0 and 3.2 mg/l given that the 1.0 mg/l test group produced approximately 5% fewer young (in terms of numbers of young produced per adult and total numbers of young produced) than the control and that no young daphnids were produced by the 3.2 mg/l test group up to day 11 where a 100 % mortality rate was observed in the parental (P1) generation.
Effects on the Filial Generation (F1)
Information on the effects of ET-344-SP on the F0 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 the 0.032, 0.10, 0.32 and 1.0 mg/ L test groups were in the same general condition as the young produced by the controls over the duration of the study.Numbers of unhatched eggs and dead young were low in all control and treatment groups surviving to maturation.
No Observed Effect Concentration
The "No Observed Effect Concentration" (NOEC) is given as 1.0 mg/l as there were no significant differences in terms of the numbers of young produced per adult when compared to the controls and no significant mortalities were observed in the parental generation (P1).

Time	EC50 (mg/ L)	95 % Confidence limits (mg/ L)
24 hours	> 3.2	-
48 hours	> 3.2	-
96 hours	3.0	2.2 -4.2
7 days	2.6	2.0 -3.4
14 days	1.8	1.0 -3.2*
21 days	1.8	1.0 -3.2 *

*Concentrations resulting in 0 and 100% immobilisation respectively.

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Daphnia magna to ET-344-SP resulted in a toxic effect at the test concentration of 3.2 mg/l between days 2 and 11. No young were produced at this test concentration as a consequence of the mortality of the adult daphnids. No impairment of reproduction was observed at the lower test concentrations of 0.032, 0.10, 0.32 and 1.0 mg/ l. The "No Observed Effect Concentration" (NOEC) is therefore given as 1.0 mg/ l.

Executive summary:

STUDY TYPE : DAPHNIA MAGNA REPRODUCTION TEST

TEST MATERIAL : ET-344-SP

Methods

A study was performed to assess the effect of the test material on the reproduction of Daphnia magna over a 21 day period. The method followed that described in the OECD Guidelines for Testing of Chemicals (1 984) No. 202 "Daphnia sp., Acute Immobilisation Test and Reproduction Test".

Procedures

Based on the results of an acute toxicity study, Daphnia magna were exposed (4 replicates of 10 daphnids per group) to an aqueous dispersion of the test material over a range of test concentrations of 0.032, 0.10, 0.32, 1.0 and 3.2 mg/l for a period of 21 days. The test solutions were renewed 3 times per week. The numbers of live and dead adult Daphnia were determined daily. The numbers of young Daphnia (live and dead) were determined at each test media renewal. The Daphnia were fed daily with a mixed algal suspension.

Results

The 14 and 21 Day EC50 (immobilisation) value, based on nominal test concentrations, for the parental Daphnia generation (P1) was calculated to be 1.8 mg/l with 95% confidence limits of 1.0-3.2 mg/l.

The 21 Day EC50 (reproduction) based on nominal test concentrations is considered to lie between 1.0 and 3.2 mg/ l given that the 1.0 mg/l test group produced approximately 5% fewer young per adult than the control group and that no young were produced by the 3.2 mg/l test group up to day 11 when a 100% mortality rate was observed in the parental (P1) generation.

The "No Observed Effect Concentration" (immobilisation and reproduction) is considered to be 1.0 mg/l on the basis that at this test concentration there were no mortalities (immobilisation) observed in the parental generation (P1) and that there were no significant differences (P>= 0.05) between the controls and the 1.0 mg/ l test group in terms of numbers of young produced per adult on days 14 and 21.

Verification of test concentrations showed that the overall mean measured test concentrations were near nominal and it was therefore considered justifiable to calculate the EC50 values in terms of nominal test concentrations only.

Description of key information

A study was performed to assess the effect of the test material on the reproduction of Daphnia magna over a 21 day period. The method followed that described in the OECD Guidelines for Testing of Chemicals (1 984) No. 202 "Daphnia sp., Acute Immobilisation Test and Reproduction Test".

The 14 and 21 Day EC50 (immobilisation) value, based on nominal test concentrations, for the parental Daphnia generation (P1) was calculated to be 1.8 mg/l with 95% confidence limits of 1.0-3.2 mg/l.

The 21 Day EC50 (reproduction) based on nominal test concentrations is considered to lie between 1.0 and 3.2 mg/ l given that the 1.0 mg/l test group produced approximately 5% fewer young per adult than the control group and that no young were produced by the 3.2 mg/l test group up to day 11 when a 100% mortality rate was observed in the parental (P1) generation.

The "No Observed Effect Concentration" (immobilisation and reproduction) is considered to be 1.0 mg/l on the basis that at this test concentration there were no mortalities (immobilisation) observed in the parental generation (P1) and that there were no significant differences (P>= 0.05) between the controls and the 1.0 mg/ l test group in terms of numbers of young produced per adult on days 14 and 21.

Verification of test concentrations showed that the overall mean measured test concentrations were near nominal and it was therefore considered justifiable to calculate the EC50 values in terms of nominal test concentrations only.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1 mg/L