Cyclopropanecarboxylic acid, 2-[1-(3,3-dimethylcyclohexyl)ethoxy]-2-methylpropyl ester

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:

23-09-2009 to 14-09-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: ISO 10706. Water quality - Determination of long term toxicity of substances to Daphnia magna Straus (Cladocera, Crustacea).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: November 2007 ; signature: March 2009

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (control), 0.0078, 0.0156, 0.0313, 0.0625, 0.125, 0.25, 0.50 mg/L nominal concentration
Arithmetic Mean Measured equivalent concentrations: < LOQ (control), 0.0076, 0.0169, 0.0338, 0.0682, 0.142, 0.292 and 0.547 mg/L
LOQ = 0.00308 mg/L or 3.08 μg/L
- Sampling method: Samples of 20 mL were taken from the designated test solutions and stock solution, and transferred to a glass tube. In order to minimise losses of the test item, the water samples were extracted on site, immediately following sampling. Samples of fresh test solutions were taken on days 0, 7 and 14 from the beakers used to prepare the solutions. Samples of aged test solutions were taken on days 2, 9 and 16 from the test vessels.
- Sample storage conditions before analysis: All samples designated for analysis were transferred to the test site for chemical analysis (detailed in the study report) on dry ice and were received at the analytical test site in good condition and placed in a freezer until analysis. The control reconstituted water sample was received in good condition and placed in a fridge until analysis. On the day of analysis, the samples were defrosted followed by shaking. The concentration of test item was determined by GC-MSD. The control reconstituted water sample was allowed to return to room temperature prior to extraction. The extraction procedure is described below.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Direct dissolution of test item in culture medium to prepared stock; serial dilution of stock to prepare test media. At the beginning of the test, and 5 more times during the test, a stock solution “S1” was prepared by dissolving 5.0 to 5.1 mg of the test item in 5 L of Medium M4 (nominal concentration 1.00 to 1.02 mg/L). This solution was ultrasonicated for 2 minutes and stirred overnight at ambient temperature in the dark. The stock solution was prepared twice per week. After the stirring period, and between use for preparation of the test item concentrations (renewal of media), S1 was stored at ambient temperature in the dark up to three days. Corresponding volumes of S1 were used to prepare the test item concentration levels by dilution with Medium M4. Each time when prepared, the volume of the stock solution was large enough to prepare all test concentration levels and all analytical samples at once. An amount of test item (5.50 g) was dispersed in 11 litres of reconstituted water and stirred. Test vessels containing 70 to 100 mL of the test solutions were placed into a temperature controlled room at test temperature for temperature adaptation. The test solutions were renewed three times a week. From chemical analysis: the mean total recovery for all measured test solutions was 108.84% of the nominal concentration. A correction of the biological results based on measured concentrations is not required. However, all study results are reported based on nominal as well as the average arithmetic mean of the measured concentrations for each individual test concentration throughout the exposure period.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution): Not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No precipitate reported.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (Straus) clone 5
- Strain/clone: See above.
- Justification for species other than prescribed by test guideline: Not applicable.
- Age at study initiation (mean and range, SD): < 24 hours
- Weight at study initiation (mean and range, SD): Not applicable.
- Length at study initiation (length definition, mean, range and SD): Not applicable.
- Stage and instar at study initiation: < 24 hours old
- Valve height at study initiation, for shell deposition study (mean and range, SD): Not applicable.
- Peripheral shell growth removed prior to test initiation: Not applicable.
- Method of breeding: Parthenogenesis
- Source: in-house laboratory cultures
- Age of parental stock (mean and range, SD): age of parents: 23 - 26 d (offspring used was not the first brood of the parental daphnids)
- Feeding during test: Yes. The daphnids were fed three times a week during the study ad libitum with algae suspension (0.1 to 0.2 mg C/daphnia /d)
- Food type: algae suspension [unicellular algal culture (Desmodesmus subspicatus)
- Amount: Approximately 0.1 - 0.2 mg carbon/daphnid/day, dependent on the age and size of the animals. Equal amounts of food were given to each daphnid.
- Frequency: Three times a week

ACCLIMATION
- Acclimation period: None. After temperature adaptation of the test solutions the daphnids were transferred into the test solutions by using an upside-down glass pipette. A one-stage randomisation procedure was used to assign the test organisms to the test solutions.
- Acclimation conditions (same as test or not): Culture conditions were equivalent to the exposure conditions.
- Type and amount of food: Not applicable.
- Feeding frequency: Not applicable.
- Health during acclimation (any mortality observed): Not applicable.

QUARANTINE (wild caught)
- Duration: Not applicable.
- Health/mortality: Not applicable.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS: Not applicable.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Remarks on exposure duration:

In accordance with the OECD TG 211 guideline.

Hardness:

Water hardness was observed to be in the range 246.5 to 250.0 mg/L as CaCO3 of the batches Medium M4 used for the test
Water hardness was observed to be in the range 250.0 to 259.0 mg/L as CaCO3 for the test media used for the test

Test temperature:

Actual: 20 +/- 2ºC (throughout test period): 19.4 to 20.9°C (manual measurement; n = 12) and 18.1 to 21.6°C (online measurement; n = 506)

pH:

7.5 to 7.6 for of the batches Medium M4 used for the test
7.5 to 7.7 (n=12 readings) for the for the test media used for the test
It was considered that there was no treatment related differences to pH. No correction to pH was employed.

Dissolved oxygen:

Dissolved Oxygen was observed to be in the range 8.4 to 9.0 mg/L (or 94 - 98% ASV) of the batches Medium M4 used for the test
Dissolved Oxygen was was observed to be in the range 8.4 to 8.9 mg/L (n=12 readings) or 94 - 99% ASV for the for the test media used for the test
It was considered that there was no treatment related differences to oxygen concentration

Salinity:

Not applicable.

Conductivity:

Conductivity was observed to be in the range 601 to 608 μS/cm of the batches Medium M4 used for the test

Nominal and measured concentrations:

Concentrations: 0 (control), 0.0078, 0.0156, 0.0313, 0.0625, 0.125, 0.25, 0.50 mg/L nominal concentration
Arithmetic Mean Measured equivalent concentrations: < LOQ (control), 0.0076, 0.0169, 0.0338, 0.0682, 0.142, 0.292 and 0.547 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL glass flasks
- Type (delete if not applicable): closed (covered to reduce evaporation) with glass lid and parafilm
- Material, size, headspace, fill volume: 7 cm x 5.4 cm ; 70 to 100 mL fill volume, i.e. 30 to 0 mL headspace
- Aeration: No. Diluent water was aerated before use.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable.
- Renewal rate of test solution (frequency/flow rate): 3 days renewal.
- No. of organisms per vessel: 1 per 100 mL vessel
- No. of vessels per concentration (replicates): 10 replicates.
- No. of vessels per control (replicates): 10 replicates.
- No. of vessels per vehicle control (replicates): Not applicable.
- Biomass loading rate: equivalent to ca. 100 mL test media per Daphnid.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt medium M4, according to OECD 202, Annex 3 and OECD 211 Annex 2
- Total organic carbon: Not reported
- Particulate matter: Not reported
- Metals: Not reported
- Pesticides: Not reported
- Chlorine: Not reported
- Alkalinity: Not reported
- Ca/mg ratio: Not reported
- Conductivity: 601 to 608 μS/cm of the batches Medium M4 used for the test
- Culture medium different from test medium: No
- Intervals of water quality measurement: Day 2, 7, 9, 14 and 16: in control and 0.50 mg/L nominal concentration test levels ; before and after renewal every other alternate measurement.

OTHER TEST CONDITIONS
- Adjustment of pH: No.
- Photoperiod: 16 h light / 8 hours dark.
- Light intensity: 7.87 to 11.25 μE m-2 s-1 (n = 5) or 623 to 870 lx (n = 5)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Reproduction and Immobility (or adverse effects including mortality)
- Fecundity: Cumulative number of living offspring per parent animal alive at the end of the test
- Immobility of Parental Daphnids
- Length of Parental Daphnids
- Observations : including reproduction and behavioural and morphological differences

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Based on preceding acute toxicity study (information in the full study report) and/or decision of the study monitor and study director.
- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study
- Test concentrations: Not applicable.
- Results used to determine the conditions for the definitive study: Not applicable.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7)

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.292 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: (95% CL. - mg/L) ; arithmetic mean measured concentration

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.547 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: (95% CL. - mg/L) ; arithmetic mean measured concentration

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 0.547 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: (95% CL. - mg/L) ; arithmetic mean measured concentration

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.547 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: (95% CL. - mg/L) ; arithmetic mean measured concentration

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.547 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

length of parental daphnids

Remarks on result:

other: (95% CL. - mg/L) ; arithmetic mean measured concentration

Details on results:

- Behavioural abnormalities: Additional observations on behavioural and/or morphological differences of the treated parent daphnids from the controls could be made occasionally across all treatments. A concentration-related pattern could not be established. This therefore was considered of no biological relevance.
- Observations on body length and weight: In P1 generation: No effect on body length was reported between control and treatment groups.
- Other biological observations: None reported of biological relevance
- Mortality of control: 1 mortality in control (i.e. 10%)
- Other adverse effects control: None reported.
- Abnormal responses: None reported of biological relevance.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The mean total recovery for all measured test solutions was 108.84% of the nominal concentration. A correction of the biological results based on measured concentrations is not required. However, all study results are reported based on nominal as well as the average arithmetic mean of the measured concentrations for each individual test concentration throughout the exposure period. It can be speculated the differences in nominal/measured test concentrations was due to possible: adsorption and biological effects and/or to any algae food/organic waste matter present in the test system.
- Effect concentrations exceeding solubility of substance in test medium: No.

Results with reference substance (positive control):

- Results with reference substance valid?: Yes.
- Mortality: See below.
- EC50/LC50: 24h-EC50 was 1.70 mg/L
- Other: The actual responses in this reference test with K2Cr2O7 are within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was between 0.6 and 2.1 mg/L. Hence, the sensitivity of this batch of test organism was in agreement with the historical data.

Reported statistics and error estimates:

The biological results, i.e. reproduction (cumulative number of neonates per surviving adult), length and immobility of adults were evaluated statistically (significance level alpha: 0.05). For evaluation of reproduction, William's test (one-sided smaller) was used. The length of the parent daphnids was evaluated by One-Way Analysis of Variance (ANOVA). Fisher`s Exact Binomial Test with Bonferroni Correction (pair-wise comparisons between treatments and control on the multiple significance level at alpha of 0.05; one-sided greater) was used to evaluate immobility of adults.
Since inhibition of the biological parameters compared to the controls was less than 20%, a calculation of Effect Concentrations (ECx) was not performed.
The statistical software package ToxRat Professional 2.10 was used.

All validity criteria were considered to be met:

1. Control mortality was ≤20% (actual 10%)

2. Dissolved oxygen was maintained ≥ 3 mg O2/L (actual ≥ 8.0 mg O2/L)

3. pH control group ≤ 1.5 deviation (actual 0.2)

5. Mean live young per surviving adult ≥ 60 after 21 days (actual minimum: > 62.0 and mean = 75.7)

6. CoV for control group ≤ 25% (actual 17.4%)

Validity criteria fulfilled:

yes

Conclusions:

The test item 21d-NOEC was 0.292 mg/L and the 21d-LOEC was 0.547 mg/L. All effects were based on arithmetic mean measured concentrations.

Executive summary:

The 21-d reproduction toxicity to Daphnia magna was carried out according to OECD TG 211 guideline under GLP. Based on the results of previously conducted acute toxicity tests, Daphnia magna were exposed (10 replicates of a single daphnid per group) to an aqueous solution of the test item over a range of nominal test concentrations of 0 (control), 0.0078, 0.0156, 0.0313, 0.0625, 0.125, 0.25, 0.50 mg/L nominal concentration for a period of 21 days. Chemical analysis indicated the mean total recovery for all measured test solutions was 108.84% of the nominal concentration and ranged from 98% to 117% of the nominal. Procedural recoveries ranged from 70 to 94% of nominally spiked concentrations in the analytical method validation. A correction of the biological results based on measured concentrations is not required. However, all study results are reported based on nominal as well as the average arithmetic mean of the measured concentrations for each individual test concentration throughout the exposure period. Equivalent Arithmetic Mean Measured concentrations were : less than the LOQ (control ; 0.00308 mg/L or 3.08 μg/L), 0.0076, 0.0169, 0.0338, 0.0682, 0.142, 0.292 and 0.547 mg/L. The test concentrations were prepared by dilution of a saturated solution of the test item. At the beginning of the test, and 5 more times during the test, a stock solution “S1” was prepared by dissolving 5.0 to 5.1 mg of the test item in 5 L of Medium M4 (nominal concentration 1.00 to 1.02 mg/L). This solution was ultrasonicated for 2 minutes and stirred overnight at ambient temperature in the dark. The stock solution was prepared twice per week. After the stirring period, and between use for preparation of the test item concentrations (renewal of media), S1 was stored at ambient temperature in the dark up to three days. Corresponding volumes of S1 were used to prepare the test item concentration levels by dilution with Medium M4. Each time when prepared, the volume of the stock solution was large enough to prepare all test concentration levels and all analytical samples at once. An amount of test item (5.50 g) was dispersed in 11 litres of reconstituted water and stirred. Test vessels containing 70 to 100 mL of the test solutions were placed into a temperature controlled room at test temperature for temperature adaptation. The test solutions were renewed three times a week. Effect parameters monitored included reproduction and immobility (or adverse effects including mortality). Specifically, fecundity: Cumulative number of living offspring per parent animal alive at the end of the test, immobility of Parental Daphnids, length of Parental Daphnids and behavioural and morphological differences. Reproduction (fecundity of parent daphnids) was influenced only at the highest test concentration (0.500 mg/L, nominal), where 16.9% inhibition was found compared with reproduction in the controls. Significant immobility of parent daphnids was not observed up to the highest concentration. Since inhibition of the biological parameters compared to the controls was less than 20%, a calculation of Effect Concentrations (ECx) was not performed. The test item 21d-NOEC (reproduction) was 0.250 mg/L based on nominal and 0.292 mg/L based on arithmetic mean measured concentrations.

Description of key information

21d-NOEC (invertebrates, reproduction) : 0.292 mg/L based on arithmetic mean measured concentrations, 21-days freshwater, OECD TG 211, 2010

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.292 mg/L

Additional information

Key study : OECD TG 211, 2010 : The 21-d reproduction toxicity to Daphnia magna was carried out according to OECD TG 211 guideline under GLP. Based on the results of previously conducted acute toxicity tests, Daphnia magna were exposed (10 replicates of a single daphnid per group) to an aqueous solution of the test item over a range of nominal test concentrations of 0 (control), 0.0078, 0.0156, 0.0313, 0.0625, 0.125, 0.25, 0.50 mg/L nominal concentration for a period of 21 days. Chemical analysis indicated the mean total recovery for all measured test solutions was 108.84% of the nominal concentration and ranged from 98% to 117% of the nominal. Procedural recoveries ranged from 70 to 94% of nominally spiked concentrations in the analytical method validation. A correction of the biological results based on measured concentrations is not required. However, all study results are reported based on nominal as well as the average arithmetic mean of the measured concentrations for each individual test concentration throughout the exposure period. Equivalent Arithmetic Mean Measured concentrations were : less than the LOQ (control ; 0.00308 mg/L or 3.08 μg/L), 0.0076, 0.0169, 0.0338, 0.0682, 0.142, 0.292 and 0.547 mg/L. The test concentrations were prepared by dilution of a saturated solution of the test item. At the beginning of the test, and 5 more times during the test, a stock solution “S1” was prepared by dissolving 5.0 to 5.1 mg of the test item in 5 L of Medium M4 (nominal concentration 1.00 to 1.02 mg/L). This solution was ultrasonicated for 2 minutes and stirred overnight at ambient temperature in the dark. The stock solution was prepared twice per week. After the stirring period, and between use for preparation of the test item concentrations (renewal of media), S1 was stored at ambient temperature in the dark up to three days. Corresponding volumes of S1 were used to prepare the test item concentration levels by dilution with Medium M4. Each time when prepared, the volume of the stock solution was large enough to prepare all test concentration levels and all analytical samples at once. An amount of test item (5.50 g) was dispersed in 11 litres of reconstituted water and stirred. Test vessels containing 70 to 100 mL of the test solutions were placed into a temperature controlled room at test temperature for temperature adaptation. The test solutions were renewed three times a week. Effect parameters monitored included reproduction and immobility (or adverse effects including mortality). Specifically, fecundity: Cumulative number of living offspring per parent animal alive at the end of the test, immobility of Parental Daphnids, length of Parental Daphnids and behavioural and morphological differences. Reproduction (fecundity of parent daphnids) was influenced only at the highest test concentration (0.500 mg/L, nominal), where 16.9% inhibition was found compared with reproduction in the controls. Significant immobility of parent daphnids was not observed up to the highest concentration. Since inhibition of the biological parameters compared to the controls was less than 20%, a calculation of Effect Concentrations (ECx) was not performed. The test item 21d-NOEC (reproduction) was 0.250 mg/L based on nominal and 0.292 mg/L based on arithmetic mean measured concentrations.