2-({[3-(isocyanatomethyl)-3,5,5-trimethylcyclohexyl]carbamoyl}oxy)ethyl prop-2-enoate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01-08-2016 to 20-08-2016

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

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

adopted April 13, 2004

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Duplicate samples from the freshly prepared test media of the only test concentration and the control were taken at the start of the test.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of the only test concentration and the control were collected at the end of the test (after 48 hours) by pouring together the contents of the test beakers of each treatment.
Additional samples of the control were taken at test start and test end without any sample treatment.
All samples were stored in a refrigerator (4 ± 4 °C), protected from light until analysis was performed.

Vehicle:

no

Details on test solutions:

The test medium was prepared by mixing 100.7 mg test item into 1007 mL test water and was carefully stirred for 48 hours in the dark to dissolve as much of the test item as possible. After cessation of mixing and a following period (1 hour) of settling to allow phase separation, the aqueous phase, i.e. the water accommodated fraction, was drawn off carefully and used as the test medium.
The test media were prepared just before introduction of the daphnids (= start of the test).

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Straus), clone 5
Age at Test Start: From 1.75 to 21.25 hours old
Sex: Female
Origin: The daphnids introduced in the test were taken from ibacon's in-house laboratory culture.
Breeding Conditions: The daphnids were bred in the laboratories of ibacon under similar temperature and light conditions as used in the test. The cultivation of the
parental daphnids was performed in Elendt M4 medium (see 6.5). The test organisms were not first brood progeny. The daphnids in the stock culture were fed at least on all working days with green algae (Desmodesmus subspicatus) freshly grown in the laboratories of ibacon.
Reference Item: For the evaluation of the quality of the Daphnia clone and the consistency of the experimental conditions, the reference item potassium
dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Acclimatisation: Was not necessary, since the test was performed in the same medium as the culturing.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

The water temperature, pH-values and dissolved oxygen concentrations were determined at test start and test end in each treatment group.
Water Temperature:
21 to 22 °C at test start;
20 to 21 °C at test end

pH:

The water temperature, pH-values and dissolved oxygen concentrations were determined at test start and test end in each treatment group.
pH-Values:
7.4 to 7.9 at test start;
7.9 to 8.0 at test end; and thus the pH-value did not vary by more than 1.5 units (see Table below)

Dissolved Oxygen Concentrations and pH-Values in the Test Media
[mg test item/L] pH O2 [mg/L] pH O2 [mg/L]
Control 7.9 9.1 8.0 8.6
45.8 7.4 8.2 7.9 8.6

Dissolved oxygen:

The water temperature, pH-values and dissolved oxygen concentrations were determined at test start and test end in each treatment group.
Dissolved Oxygen Concentration:
8.2 to 9.1 mg/L at test start;
8.6 mg/L at test end (see Table below)

Dissolved Oxygen Concentrations and pH-Values in the Test Media
[mg test item/L] pH O2 [mg/L] pH O2 [mg/L]
Control 7.9 9.1 8.0 8.6
45.8 7.4 8.2 7.9 8.6

Nominal and measured concentrations:

At the start of the test 47 % of the nominal test concentration was found (average of duplicate samples of the only test concentration). After 48 hours test duration, 45 % of the nominal value was determined (average of duplicate samples of the only test concentration). During the test the daphnids were exposed to a mean of 46 % of nominal. Therefore, all reported results refer to mean measured concentrations.

Details on test conditions:

Course of the Test
Introduction of Individuals: 20 daphnids per control and test concentration, divided into 4 groups of 5 animals, each group in 60 mL test medium
Exposure Time: 48 hours
Test Procedure: A static test was performed.

Test Conditions
Light Regime: 16 h light : 8 h dark
Light Intensity: The light intensity was 640 to 780 lux (measured once during the test).

Test Parameters
Immobility: The mobility of the daphnids was determined by visual observation after 24 and 48 hours. Those animals that are not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile (even if they could still move their antennae).



Test Water
Reconstituted Water
(Elendt "M4"):
Analytical grade salts and additives were added at the following nominal concentrations in
deionised water (conductivity < 5 µScm-1):

Main Compounds Trace Elements
CaCl2·2 H2O 293.80 mg/L H3BO3 2860 µg/L Thiamin HCl 75.0 µg/L
MgSO4·7 H2O 123.30 mg/L MnCl2·4 H2O 361 µg/L Cyanocobalamin (B12) 1.0 µg/L
KCl 5.80 mg/L LiCl 306 µg/L Biotin (B6) 0.75 µg/L
NaHCO3 64.80 mg/L RbCl 71 µg/L
Na2SiO3·9 H2O 10.00 mg/L SrCl2·6 H2O 152 µg/L
NaNO3 0.27 mg/L NaBr 16 µg/L
KH2PO4 0.14 mg/L Na2MoO4·2 H2O 63 µg/L
K2HPO4 0.18 mg/L CuCl2·2 H2O 17 µg/L
ZnCl2 13 µg/L
CoCl2·6 H2O 10 µg/L
KI 3.3 µg/L
Na2SeO3 2.2 µg/L
NH4VO3 0.6 µg/L
Na2EDTA·2 H2O 2.5 mg/L
FeSO4·7 H2O 1.0 mg/L
Water Hardness: 2.5 mmol/L (= 250 mg/L) as CaCO3
Alkalinity: 0.9 mmol/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

> 45.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 45.8 mg/L

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 45.8 mg/L

Details on results:

Influence of test item on the Mobility of Daphnia magna

Mean Measured No. of Daphnia No. of immobilised % of immobilised
Concentration tested Daphnia after Daphnia after
[mg test item/L] 24 h 48 h 24 h 48 h

Control 20 0 0 0 0
45.8 20 0 0 0 0





Summary of Analytical Results

sample description % of RSD
[mg/L] nominal (1) [%] n

Control n.a. n.a. 4
100 46 2.09 4

(1) mean value of all measured samples per treatment group
RSD: relative standard deviation per treatment group n: number of analysed samples
n.a.: not applicable

Results with reference substance (positive control):

Results of the most recent Test with the Reference Item Potassium dichromate performed in January 2016 (88295220), see table " any other information on results"

Results of the most recent Test with the Reference Item Potassium dichromate performed in January 2016 (88295220)

Nominal Concentration	% of immobilised daphnids after
[mg test item/L]	24 hours	48 hours
Control	0	0
0.125	0	0
0.25	0	0
0.5	0	0
1.0	10	55
2.0	75	100
EC50[mg/L]:	1.58	0.986
95 % CI [mg/L]:	1.32 - 1.92	n.d.
EC20[mg/L]:	1.17	0.897
95 % CI [mg/L]:	0.856 - 1.39	n.d.
EC10[mg/L]:	1.00	0.854
95 % CI [mg/L]:	0.656 - 1.22	n.d.
NOEC [mg/L]:	0.5	0.5
LOEC [mg/L]:	1.0	1.0
Values refer tonominaltest concentrations CI: Confidence interval n.d.: not determinable NOEC and LOEC were determined directly from the raw data.

Validity criteria fulfilled:

yes

Conclusions:

The 48-hour NOEC was determined to be ≥ 45.8 mg test item/L and / or its degradation products.
The 48-hour LOEC was determined to be > 45.8 mg test item/L and / or its degradation products and the 48-hour EC50 value was determined to be > 45.8 mg test item/L and / or its degradation products.

Executive summary:

The purpose of this study was to determine the influence of the test item on the mobility of Daphnia magna. Young daphnids (< 24 hours old) were exposed in a static test for 48 hours to test water containing the test item at the concentration of nominal  100 mg  test  item/L.

This  limit  test  was  performed  in compliance with the test guidelines to demonstrate that the test item has no toxic effect on daphnids up to at least this concentration.

The method of application and the test species Daphnia magna are recommended by the test guidelines.

Conclusion

The toxic effect of the test item to Daphnia magna was assessed in a static limit test.

The 48-hour NOEC was determined to be ≥ 45.8 mg test item/L and / or its degradation products.

The 48-hour LOEC was determined to be > 45.8 mg test item/L and / or its degradation products and the 48-hour EC50 value was determined to be > 45.8 mg test item/L and / or its degradation products.

Description of key information

The purpose of this study was to determine the influence of the test item on the mobility of Daphnia magna. Young daphnids (< 24 hours old) were exposed in a static test for 48 hours to test water containing the test item at the concentration of nominal  100 mg  test  item/L.

This  limit  test  was  performed  in compliance with the test guidelines to demonstrate that the test item has no toxic effect on daphnids up to at least this concentration.

The method of application and the test species Daphnia magna are recommended by the test guidelines.

Conclusion

The toxic effect of the test item to Daphnia magna was assessed in a static limit test.

The 48-hour NOEC was determined to be ≥ 45.8 mg test item/L and / or its degradation products.

The 48-hour LOEC was determined to be > 45.8 mg test item/L and / or its degradation products and the 48-hour EC50 value was determined to be > 45.8 mg test item/L and / or its degradation products.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

> 45.8 mg/L