6-{4-[(1E)-3-methoxy-3-oxoprop-1-en-1-yl]phenoxy}hexyl methacrylate

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:

Nov. 2016 - Feb. 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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: ROC-601
CAS Number: 439661-46-8
Appearance: White crystalline powder
Purity: 99.5 %
Storage: at room temperature, avoid exposure to light
Lot no: S23PSG0816
Expiration date: June 28, 2018

Analytical monitoring:

yes

Details on sampling:

For determination of the test item concentration, four replicate samples were taken from the test item treatment group at the start and end of each renewal period of the test. Two control samples were analysed on each analytical occasions.
The analysis of the test item ROC-601was performed by the analytical laboratory of TOXI-COOP ZRT. according to the results of the analytical method validation (Study number: 888-100-1898) by HPLC method with UV detection.

Vehicle:

no

Details on test solutions:

For preparation of test solution a supersaturated solution (100 mg/L nominal loading) was prepared by adding an amount of 0.05 g test item in 500 mL test medium (ISO medium). This supersaturated solution was shaken for 20 hours and thereafter the non-dissolved test material was removed by filtration through a 0.22 µm membrane filter to obtain the saturated test solution. The test solution was freshly prepared in the testing laboratory just before introduction of the test animals (start of each renewal period of the experiment).
Composition of ISO medium:
Substance:Nominal concentration of the stock solutions (g/L)
CaCl2 x 2 H2O: 11.76 g/L
MgSO4 x 7 H2O: 4.93 g/L
NaHCO3: 2.59 g/L
KCl: 0.23 g/L

Test organisms (species):

Daphnia magna

Details on test organisms:

Species and strain: Daphnia magna (Straus)
Origin: Laboratory of Hydrobiology (Central Agricultural Office, Directorate of Plant-, and Soil Protection), 2100-Gödöllő, Kotlán S. u. 3. Hungary
Breeding: The Daphnia are bred in the Laboratory of TOXI-COOP ZRT.
Number of animals: Twenty animals in both groups (concentration and untreated control), divided into 4 concurrent batches (5 animals per batch).
Age of animals: Less than 24 h old at the beginning of the test.
Sex: Female
Animal health: Apparently healthy animals were used in this test with a known history
Acclimatization: Test animals were bred under similar (or the same) conditions as that used during the exposure period (holding water, temperature, background colour etc.), therefore additional acclimatisation before the test was not necessary. Brood daphnids were maintained in dilution water at the test temperature for at least 48 hours prior to the start of the test.
Food and Feeding: The Daphnia were fed with concentrated algal suspension of Raphidocelis subcapitata during the holding. Test animals were not fed during the exposure.
Reconstituted water (ISO medium, according to OECD 202) was used as dilution water in the experiment. Separate stock solutions of individual substances were first prepared in deionised water (prepared in TOXI-COOP ZRT. by MILLIPORE ELIX 3 water purification system). The ISO medium was prepared by adding 25 mL from each of four stock solutions to one litre deionised water.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Remarks on exposure duration:

Standard exposure time according to OECD guideline

Post exposure observation period:

None

Hardness:

The measured hardness of ISO medium was 237 mg/L.

Test temperature:

19.8 – 20.4 °C measured in the test vessels during the test

pH:

The pH was in the range of 7.77 – 8.02 during the test

Dissolved oxygen:

7.56 – 8.37 mg/L during the test

Conductivity:

not measured

Nominal and measured concentrations:

Based on the results of the non-GLP Preliminary Range-Finding Test (see above), the main test included a nominal concentration of 100 mg/L (limit test) and a concurrent control group. The exposure concentration was calculated as the geometric mean of the start and end values of each renewal period and determined to be 1.97 µg/L analytically.

Details on test conditions:

The test item is unstable in light; therefore, the experiment was carried out in complete darkness.

Reference substance (positive control):

yes

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

> 1.97 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Remarks on result:

other: equivalent to > 100 mg/L nominal

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 1.97 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Remarks on result:

other: equivalent to > 100 mg/L nominal

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1.97 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Remarks on result:

other: equivalent to > 100 mg/L nominal

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 1.97 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Remarks on result:

other: equivalent to > 100 mg/L nominal

Details on results:

Validity of the Study
Immobilisation was not observed in the control group and the dissolved oxygen concentration at the end of the test in control and test vessels was more than 3 mg/L in all cases. All validity criteria were within acceptable limits and therefore the study can be considered as valid.
Analytical Results
A saturated test item solution (limit concentration) and a concurrent control were included in the main test. The concentration of the test item was analytically determined at the start and at the end of each renewal period of the experiment.
The test item was not detected in the untreated control group.
In the treated group mean of the measured concentrations was 1.89 µg/L at the start and 0.98 µg/L at the end of the first renewal period. In the second renewal period the measured test item concentrations were 3.56 µg/L at the start and 2.29 µg/L at the end. The exposure concentration was calculated as the geometric mean of the start and end values of each renewal period and determined to be 1.97 µg/L. This concentration was considered as the saturation concentration in the test medium (equivalent to 100 mg/L nominal concentration).
Biological Results
There was no immobilisation observed in twenty daphnids exposed to either test item treated or control group. In addition to immobility, no abnormal behaviour or appearance of test animals was detected.

Results with reference substance (positive control):

For the evaluation of the quality of the Daphnia clone and the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions. The date of the last study (Study Code: 392-202-1922) with reference item Potassium dichromate was 20 - 21 September 2016.
The 24h EC50 value was determined to be 1.57 mg/L (95% conf. limits: 1.31 – 1.79 mg/L).
Name and data of Reference Item
Name: Potassium dichromate (K2Cr2O7)
Batch No.: MKBN3524V
Description: Orange crystalline powder
Expiry Date: 04 March, 2018
Storage: Room temperature
Supplier: SIGMA-ALDRICH

Reported statistics and error estimates:

A limit test was performed and toxic effects were not observed. Therefore, statistical analysis was not necessary. The NOEC, LOEC and ECx values of the test item were determined directly from the raw data.

ROC-601concentrations measured in the test solutions

Nominal Concentration  (mg/L)	Measured concentrationswith 95% confidence interval, µg/L
	1^stRenewal Period		2^ndRenewal Period
	Start Nov. 09, 2016	End Nov. 10, 2016	Start Nov. 10, 2016	End Nov. 11, 2016
100	1.95	0.96	3.59	2.55
	2.00	1.06	3.81	2.09
	1.76	0.87	3.33	2.40
	1.85	1.01	3.53	2.10
Meanwith 95% confidence interval, (µg/L)	1.89 ± 0.17	0.98 ± 0.13	3.56 ± 0.31	2.29 ± 0.37

Immobilization of the test animals

Test Group	Replicate	Number of treated animals	Number of immobilised animals
			24 h	48 h
Control	1	5	0	0
	2	5	0	0
	3	5	0	0
	4	5	0	0
Saturated test concentration (1.97 µg/L measured)	1	5	0	0
	2	5	0	0
	3	5	0	0
	4	5	0	0

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of this study, the test item ROC-601 had no toxic effect at aquatic saturation (i.e. water accommodated fraction, WAF) on Daphnia magna; the EC10, EC20, EC50 results and the LOEC are higher than the solubility level of the test item in the test medium (1.97 µg/L on average), which corresponds to a nominal concentration of 100 mg/L.

Executive summary:

Following a limit test in which daphnia were exposed to the test item for 48 hours at the limit of its solubility in the test medium (i.e.water accommodated fraction, WAF) indicating that the test item has no influence on the mobility of Daphnia up to at least the saturation concentration (equivalent to 100 mg/L nominal concentration), a full study was performed.

The substance ROC-601 was assessed using Daphnia magna in a 48-hour acute immobilisation test. Young Daphnia were exposed to aqueous test media containing the test item for 48 hours in a semi-static test.

The quantification of the test item ROC-601 was performed by a previously validated analytical method. Samples were taken from the test concentration and the control at the start and at the end of each renewal period of the experiment and analysed by HPLC methodwith UV detection. The determined exposure concentration of the test item was 1.97 µg/L which was calculated as the geometric mean of the measured start and end concentrations.

Twenty Daphnia (divided into 4 concurrent batches) were tested each, exposed to the saturated test concentration and in the control (ISO medium). Environmental conditions were recorded and remained within acceptable ranges.

The immobilisation of the test animals was recorded 24 and 48 hours after treatment. There was no immobilisation in twenty daphnids exposed to either test item treated or control group and no abnormal behaviour or appearance of test animals was detected.

All validity criteria were met and therefore the study can be considered as valid.

In conclusion the test item ROC-601 had no toxic effect at aquatic saturation (i.e.water accommodated fraction, WAF) on Daphnia magna; the EC_10,EC_20,EC₅₀ results and the LOEC are higher than the solubility level of the test item in the test medium (> 1.97 µg/L), which corresponds to a nominal concentration of 100 mg/L.

Description of key information

The test item ROC-601 had no toxic effect at aquatic saturation (i.e.water accommodated fraction, WAF) on Daphnia magna; the EC_10, EC_20, EC₅₀ results and the LOEC are higher than the solubility level of the test item in the test medium (> 1.97 µg/L), which corresponds to a nominal concentration of 100 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L