2,2-bis[(allyloxy)methyl]butyl (3-{[({2,2-bis[(allyloxy)methyl]butoxy}carbonyl)amino]methyl}-3,5,5-trimethylcyclohexyl)carbamate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 September 2017 to 09 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures.

Version / remarks:

OECD series on testing and assessment number 23, 2000.

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. In addition, the glass wool containing the undissolved residue from the highest test concentration was kept for possible analysis.
- Frequency: At t=0 h and t=48 h
- Volume: 1.0 mL from the approximate centre of the test vessels.
- Sample storage conditions before analysis: Samples were stored in a freezer (¿-15 °C) until analysis at the analytical laboratory of the Test Facility.
- At the end of the exposure period, the replicates were pooled at each concentration before sampling.
- Additionally, reserve samples of 1.0 mL were taken for possible analysis. If not used, these samples were stored in a freezer (¿-15 °C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- The batch of test material tested was a clear colourless viscous liquid and not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test material.
- Preparation of test solutions started with loading rates individually prepared at 1.0, 10 and 100 mg/L. A three-day period of magnetic stirring was applied to ensure maximum dissolution of the test material in medium. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning through glass wool and used as test concentrations. Due to an indicated sensitivity of the test material towards light, the preparation of test solutions was performed under dimmed light conditions and glassware was wrapped in aluminium foil. All test solutions were clear and colorless at the end of the preparation procedure.
- Any residual volumes were discarded.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain/clone: Straus, 1820
- Source: In-house laboratory culture with a known history. At least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions. Daphnids originated from a healthy stock, 2nd to 5th brood.
- Age: For the test selection of young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old.
- Feeding during test: No

ACCLIMATION
- Acclimation period: Each batch is started with newborn daphnids, i.e. less than 3 days old, by placing about 250 into 5 litres of medium in an all-glass culture vessel. The maximum age of the cultures is 4 weeks. After 7 days of cultivation, half of the medium is renewed twice a week.
- Acclimation conditions (same as test or not): Yes. Daphnids were kept in medium M7 at 18 to 22 °C.
- Type and amount of food: A suspension of fresh water algae.
- Feeding frequency: Daily
- Health during acclimation: Daphnia showed no signs of stress such as mortality >20 %, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

180 mg/L expressed as CaCO3.

Test temperature:

19-20 °C

pH:

7.9-8.1

Dissolved oxygen:

8.2-9.9 mg/L

Nominal and measured concentrations:

Nominal: 1.0, 10 and 100 mg/L (WAF loading rate)
Measured: 0.41 mg/L (average exposure concentration (geometric mean) of 100 mg/L WAF)

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL
- Material, size, fill volume: All-glass, 50 mL of solution.
- Aeration: No aeration of the test solutions.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4 for the highest concentration, 2 per intermediate concentration.
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The following chemicals are dissolved in tap water purified by Reverse Osmosis (RO-water):
Adjusted ISO medium: Macro salts: CaCl2.2H2O 211.5 mg/L, MgSO4.7H2O 88.8 mg/L, NaHCO3 46.7 mg/L and KCl 4.2 mg/L.

Medium M7: trace elements, macronutrients and vitamins are added to freshly prepared ISO medium to reach the following concentrations:
Trace elements: B 0.125 mg/L, Fe 0.05 mg/L, Mn 0.025 mg/L, Li, Rb and Sr 0.0125 mg/L, Mo 0.0063 mg/L, Br 0.0025 mg/L, Cu 0.0016 mg/L, Zn 0.0063 mg/L, Co and I 0.0025 mg/L, Se 0.0010 mg/L, V 0.0003 mg/L and Na2EDTA.2H2O 2.5 mg/L.
Macro nutrients: Na2SiO3.9H2O 10.0 mg/L, NaNO3 0.27 mg/L, KH2PO4 0.14 mg/L and K2HPO4 0.18 mg/L.
Vitamins: Thiamine 75.0 ¿g/L, B12 1.0 ¿g/L and Biotin 0.75 ¿g/L.

- Culture medium different from test medium: Daphnids were cultured in medium M7 and the test carried out in adjusted ISO medium.
- Intervals of water quality measurement: At the beginning and at the end of the test for the highest concentration and the control, pH and dissolved oxygen were determined. Temperature of medium was monitored continuously in a temperature control vessel, beginning at the start of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Test was performed in the dark.

EFFECT PARAMETERS MEASURED
- Immobility (including mortality) was determined at 24 hours and at 48 hours.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.41 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

ACCEPTABILITY OF THE TEST
- In the control, no daphnids became immobilised or showed other signs of disease or stress, for example discoloration or unusual behaviour such as trapping at the surface of the medium.
-The results of measurement of pH and oxygen remained within the limits prescribed by the study plan (pH: 6-9, not varying by more than 1.5 units; oxygen: ¿ 3 mg/L at the end of the test).
-The temperature continuously measured in a temperature control vessel varied between 19 and 20 °C during the test, and complied with the requirements as laid down in the study plan (18-22 °C, constant within 2 °C).


MEASURED CONCENTRATIONS
- Samples taken from the WAF prepared at a loading rate of 100 mg/L were analysed and showed a concentration of 0.68 mg/L at the start of the test. This concentration decreased to 35 % of the initial concentration at the end of the test. Based on this result, the average exposure concentration (geometric mean) was calculated to be 0.41 mg/L and used for the determination of the EC50 value.
It should be noted that a small response was detected in the control. These samples were likely contaminated during the course of sample treatment.

IMMOBILITY
- No immobility was observed at any of the test concentrations and the control.

Results with reference substance (positive control):

- The 24h-EC50 was 0.59 mg/L with a 95 % confidence interval between 0.52 and 0.68 mg/L.
- The 48h-EC50 was 0.41 mg/L with a 95 % confidence interval between 0.34 and 0.46 mg/L.
- 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 0.3 and 1.0 mg/L. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at the testing facility.

Reported statistics and error estimates:

No EC50 could be calculated because the test material proved to be non-toxic (EC50 > maximum soluble concentration tested).

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the 48h-EC50 for Daphnia magna exposed to the test material exceeded the maximum solubility of the test material in test medium, i.e., exceeded an average concentration of 0.41 mg/L.

Executive summary:

The potential of the test material to cause aquatic toxictity to invertebrates was investigated in accordance with the standardised guideline OECD 202, under GLP conditions.

The test material was evaluated for its ability to generate acute toxic effects on the mobility of Daphnia magna during an exposure period of 48 hours and, if possible, to determine the EC50 at 24 and 48 hours of exposure.

A combined limit/range-finding test was performed. Water Accommodated Fractions (WAFs) were individually prepared at loading rates of 1.0, 10 and 100 mg/L and used as test concentrations.

Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to a WAF prepared at a loading rate of 100 mg/L, in a limit test. In addition ten daphnids per group (5 per replicate, duplicate) were exposed to WAFs prepared at loading rates of 1.0 and 10 mg/L in the combined range-finding test. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

Due to an indicated sensitivity of the test material towards light, the preparation of test solutions was performed under dimmed light conditions and glassware was wrapped in aluminium foil.

No immobility was observed at any of the test concentrations and the control.

Samples taken from the WAF prepared at a loading rate of 100 mg/L were analysed and showed an initial concentration of 0.68 mg/L. This concentration decreased to 35 % of initial at the end of the test. Based on this result, the average exposure concentration (geometric mean) was calculated to be 0.41 mg/L and this concentration was used for the determination of the EC50 value.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

Under the conditions of this study, the 48h-EC50 for Daphnia magna exposed to the test material exceeded the maximum solubility of the test material in test medium, i.e., exceeded an average concentration of 0.41 mg/L.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

3 September 2009 to 14 October 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

yes

Remarks:

24 hour duration

Qualifier:

according to guideline

Guideline:

other: State Environmental Protection Administration of China. The Guidelines for the Testing of Chemicals, 202 Daphnia sp. 24h EC55 Acute immobilization Test

Version / remarks:

First edition. Beijing: China Environmental Science Press. 2004. 182-187

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: the test solution with nominal concentration of 100 mg/L and was analysed at the start and the end of the test.
- Test solutions were centrifuged (12000 rpm) for 5 minutes, the supernatant was collected and was centrifuged (12000 rpm) for 5 minutes again. The final solution was stored at -20°C until analysis.

Vehicle:

yes

Remarks:

tetrahydrofuran

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 2000 mg test substance was dissolved in 2000 mg tetrahydrofuran. Then the volume of solution was measured by a 10 mL graduated flask and was 3.6 mL. The nominal concentration of the stock solution was 555.6 g/L.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Age of parental stock: ¿ 24 h
- Feeding during test: no

ACCLIMATION
- Acclimation period: 3 days

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

24 h

Test temperature:

20 ± 2 °C

pH:

7.2 to 7.7

Dissolved oxygen:

7.59 to 8.02 mg/L

Nominal and measured concentrations:

Nominal: 100 mg/L
Measured: 1.82 µg/L (start of the test), 1.80 µg/L (end of the test)

Details on test conditions:

TEST SYSTEM
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Preparation the test water with the following solutions:
CaCl2.2H2O solution: 11.76 g CaCl2.2H2O was dissolved in deionised water and diluted with the water to 1 L.
MgSO4.7H2O solution: 4.93 g MgSO4.7H2O was dissolved in deionised water, and diluted with the water to 1 L.
NaHCO3 solution: 2.59 g NaHCO3 was dissolved in deionised water, and diluted with the water to 1 L.
KCl solution: 0.23 g KCl was dissolved in deionised water, and diluted with the water to 1 L.
25 mL of each solution was mixed, and diluted with the deionised water to 1 L. If necessary, pH of test water was adjusted to 7.8 ± 0.2 by HCI and NaOH.

OTHER TEST CONDITIONS
- Adjustment of pH: yes, if necessary, pH of test water could be adjusted to 7.8 ± 0.2 by HCI and NaOH.
- Photoperiod: 16 h / 8 h

EFFECT PARAMETERS MEASURED:
- Overview: responses of daphnids were frequently observed during the test, and the dead daphnids were removed as soon as possible. Immobilities including mortalities in each vessel were recorded at 24 h.
-Temperature: temperature of the controls and each test group were measured at the start and the end of the test.
-pH: pH of the controls and each test group were measured at the start and the end of the test.
- Dissolved oxygen: dissolved oxygen of the controls and each test group were measured at the start and the end of the test.
- Analysis of test concentrations: the test solution with nominal concentration of 100 mg/L and was analysed at the start and the end of the test.

VEHICLE CONTROL PERFORMED: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

RESULTS
- Dissolved oxygen was 7.59 to 8.02 mg/L
- Temperature was 18.9 to 20.1°C
- pH was 7.2 to 7.7
- Daphnia magna immobility of blank control and solvent control was 0%
- Daphnia magna mortality of test substance group was 5% at end of the test
- Analysis of the test solution with nominal concentration of 100 mg/L showed that the measured concentration of the test substance was 1.82 µg/L at 0 h and was 1.80 µg/L at 24 h.

QUALITY ASSURANCE
- At the end of the test, the dissolved oxygen concentrations of the test solutions were higher than 3 mg/L.
- At the end of the test, immobility of Daphnia in the control was lower than 10%.
- The 24h-EC50 of potassium dichromate was within the range of 0.6 mg/L to 2.0 mg/L.

Results with reference substance (positive control):

- KPotassium dichromate was used as a reference substance to determine the sensitivity of Daphnia magna. A reference substance group was set up with concentrations of 0.5, 0.81, 1.32, 2.15 and 3.5 mg/L and a blank control. 20 daphnids were exposed to each concentration.
- The test result of Daphnia magna sensitivity revealed that 24h-EC50 of potassium dichromate was 1.87 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the 24h EC50 of the test material (based on the nominal concentration) of Daphnia magna was 100 mg/L.

Executive summary:

The acute toxicity of the test material to Daphnia magna was investigated in accordance with the standardised guidelines "State Environmental Protection Administration of China. The Guidelines for the Testing of Chemicals 202 Daphnia sp. 24h EC50 Acute Immobilization Test" and OECD 202 and under GLP conditions. The limit test was conducted with the nominal concentration of 100 mg/L, exposing twenty daphnids for a test period of 24 hours. Responses of daphnids were frequently observed during the test, and the dead daphnids were removed as soon as possible. Immobilities including mortalities in each vessel were recorded at 24 hours. Analysis of the test solution with nominal concentration of 100 mg/L showed that the measured concentration of the test material was 1.82 µg/L at 0 h and was 1.80 µg/L at 24 h. At the end of the test, the dissolved oxygen concentrations of the test solutions were higher than 3 mg/L, immobility of Daphnia in the control group was lower than 10% and the 24 h EC50 of potassium dichromate (the reference substance) was within the range of 0.6 mg/L to 2.0 mg/L so that the validity criteria of the test were met. Under the conditions of the study, the 24 h EC50 of the test material (based on the nominal concentration) for Daphnia magna was 100 mg/L.

Description of key information

Key Study: Augusiak (2018)

Under the conditions of this study, the 48h-EC50 for Daphnia magna exposed to the test material exceeded the maximum solubility of the test material in test medium, i.e., exceeded an average concentration of 0.41 mg/L.

Additional information

Key study: Augusiak (2018)

The potential of the test material to cause aquatic toxictity to invertebrates was investigated in accordance with the standardised guideline OECD 202, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was evaluated for its ability to generate acute toxic effects on the mobility of Daphnia magna during an exposure period of 48 hours and, if possible, to determine the EC50 at 24 and 48 hours of exposure.

A combined limit/range-finding test was performed. Water Accommodated Fractions (WAFs) were individually prepared at loading rates of 1.0, 10 and 100 mg/L and used as test concentrations.

Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to a WAF prepared at a loading rate of 100 mg/L, in a limit test. In addition ten daphnids per group (5 per replicate, duplicate) were exposed to WAFs prepared at loading rates of 1.0 and 10 mg/L in the combined range-finding test. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

Due to an indicated sensitivity of the test material towards light, the preparation of test solutions was performed under dimmed light conditions and glassware was wrapped in aluminium foil.

No immobility was observed at any of the test concentrations and the control.

Samples taken from the WAF prepared at a loading rate of 100 mg/L were analysed and showed an initial concentration of 0.68 mg/L. This concentration decreased to 35 % of initial at the end of the test. Based on this result, the average exposure concentration (geometric mean) was calculated to be 0.41 mg/L and this concentration was used for the determination of the EC50 value.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

Under the conditions of this study, the 48h-EC50 for Daphnia magna exposed to the test material exceeded the maximum solubility of the test material in test medium, i.e., exceeded an average concentration of 0.41 mg/L.

Supporting study: Chengfang (2009)

The acute toxicity of the test material to Daphnia magna was investigated in accordance with the standardised guidelines "State Environmental Protection Administration of China. The Guidelines for the Testing of Chemicals 202 Daphnia sp. 24 h EC50 Acute Immobilization Test" and OECD 202 and under GLP conditions. The limit test was conducted with the nominal concentration of 100 mg/L, exposing twenty daphnids for a test period of 24 hours. Responses of daphnids were frequently observed during the test, and the dead daphnids were removed as soon as possible. Immobilities including mortalities in each vessel were recorded at 24 hours. Analysis of the test solution with nominal concentration of 100 mg/L showed that the measured concentration of the test material was 1.82 µg/L at 0 h and was 1.80 µg/L at 24 h. At the end of the test, the dissolved oxygen concentrations of the test solutions were higher than 3 mg/L, immobility of Daphnia in the control group was lower than 10 % and the 24 h EC50 of potassium dichromate (the reference substance) was within the range of 0.6 mg/L to 2.0 mg/L so that the validity criteria of the test were met. Under the conditions of the study, the 24 h EC50 of the test material (based on the nominal concentration) for Daphnia magna was 100 mg/L.