Reaction mass of ethyl 2,6,6-trimethylcyclohexa-1,3-diene-1-carboxylate and ethyl 2,6,6-trimethylcyclohexa-2,4-diene-1-carboxylate and ethyl 6,6-dimethyl-2-methylenecyclohex-3-enecarboxylate

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:

14 November - 19 December 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)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

ECT (Date of inspection: 02 February 2015 and 09 March 2015; Signed on: 12 June 2015) CIP (Date of inspection: 13 June 2016; Signed on: 08 September 2016)

Analytical monitoring:

yes

Details on sampling:

Samples of stock solution and test solutions were taken to determine the actual test item concentrations in comparison to the nominally applied concentrations. Control and test solutions were sampled in duplicate using volumetric glass pipettes. The duplicate samples were kept separately as a reserve. The nominal volume per sample was 20 mL. The volume of each sample was recorded.

After sampling and before shipment, all samples were stored in amber glass bottles in the dark at a temperature of ≤ -18 °C. A record was kept for each sample. Sampling was done at the start of exposure and after 48 hours of exposure.

Samples of untreated test medium were provided to the analytical laboratory in order to determine the recovery of the analytical method with the specific test medium. Samples were transferred to the test site for chemical analysis under the required storage conditions.

Analysis was performed according to an analytical method validated by the test site for chemical analysis.

Details on test solutions:

A series of concentrations of the test item in aqueous solution was prepared, and the test organisms were exposed to these concentrations, as well as to controls without the test item, for a period of 48 hours. During the test period the test solutions were not aerated and not renewed. The following concentrations in a geometrical series (spacing factor: 2.8) were tested in the definitive test: 0.81, 2.28, 6.38, 17.9 and 50.0 mg test item/L.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia
- Strain/clone: Daphnia magna STRAUS (clone M10)
- Age at study initiation: less than 24 h
- Source: cultured at ECT Oekotoxikologie GmbH; organisms were originally supplied by KU Leuven, Belgium.
- Age of parental stock: less than 35 d
- Feeding during test: less than 35 d

ACCLIMATION
- Type and amount of food: Algae (Desmodesmus subspicatus), instant baker’s yeast suspension, TetraMin® suspension
- Feeding frequency: 4 times per week

Test type:

static

Water media type:

other: Elendt medium M4

Limit test:

no

Total exposure duration:

48 h

Hardness:

250 mg/L CaCO3

Test temperature:

20.5 - 21.7 °C

pH:

7.5-7.7

Dissolved oxygen:

7.9 - 8.2 mg/L

Conductivity:

670 μS/cm

Nominal and measured concentrations:

Nominal concentrations: 0.81, 2.28, 6.38, 17.9 and 50.0 mg test item/L
The nominal concentrations mentioned above correspond to the geometric mean measured concentrations of: 0.534, 1.76, 4.61, 12.2 and 34.3 mg test item/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 60-mL glass beakers
- Volume of solution:50-60 mL
- Aeration: None
- Renewal rate of test solution (frequency/flow rate): None (static exposure)
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4 - No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Composition of Medium:

Trace elements:
H3BO3: 2.86 mg/L
MnCl2 × 4 H2O: 0.361 mg/L
LiCl: 0.306 mg/L
RbCl: 0.071 mg/L
SrCl2 × 6 H2O: 0.152 mg/L
NaBr: 0.016 mg/L
NaNO3: 0.274 mg/L
Na2MoO4 × 2 H2O: 0.063 mg/L
CuCl2 × 2 H2O: 0.017 mg/L
ZnCl2: 0.013 mg/L
CoCl2 × 6 H2O: 0.010 mg/L
KI: 0.0033 mg/L
Na2SeO3: 0.0022 mg/L
NH4VO3: 0.00058 mg/L
Na2EDTA × 2 H2O: 2.5 mg/L
FeSO4 × 7 H2O: 1.0 mg/L

Macronutrients:
CaCl2 × 2 H2O: 293.8 mg/L
MgSO4 × 7 H2O: 123.3 mg/L
KCl: 5.8 mg/L
NaHCO3: 64.8 mg/L
Na2SiO3 × 9 H2O: 10.0 mg/L
KH2PO4: 0.143 mg/L
K2HPO4: 0.184 mg/L

Vitamins:
Thiamine hydrochloride: 0.075 mg/L
Cyanocobalamin: 0.001 mg/L
Biotine: 0.00075 mg/L

- Alkalinity: 0.8 mmol/L HCO3
- Conductivity: 670 μS/cm
- Intervals of water quality measurement: At start and end of the test

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod: 16 h light : 8 h dark
- Light intensity: 50 to 1000 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Immobility

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Results used to determine the conditions for the definitive study: 0.81, 2.28, 6.38, 17.9 and 50.0 mg test item/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (K2Cr2O7)

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

20.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limit

Remarks:

13.9-24.9 mg/L

Duration:

48 h

Dose descriptor:

other: EC20

Effect conc.:

23 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limit

Remarks:

17.0-28.3 mg/L

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

29.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limit

Remarks:

23.9-37.4 mg/L

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

13.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limit

Remarks:

9.45-17.0 mg/L

Duration:

48 h

Dose descriptor:

other: EC20

Effect conc.:

15.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limit

Remarks:

11.6-19.3

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

20.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limit

Remarks:

16.3-25.6

Results with reference substance (positive control):

The reference test with potassium dichromate resulted in an EC50(24 h) of 1.68 mg/l..

According to the OECD guideline No. 202 (OECD, April 2004) EC50(24 h)-values for potassium dichromate obtained from different laboratories should range between 0.6 mg/l and 2.1 mg/l.
The toxicity of the reference item is within this range. Therefore the results of this reference test are acceptable and the test conditions are reliable and the sensitivity of the test system could be demonstrated.

Biological Results

A summary of the immobility data for Daphnia magna are shown in the following table.

Table 6.1.3/1: Cumulative number of immobilised Daphnia magna after 24 and 48 h.

Nominal concentration	total number of introduced daphnids	total number of immobilised daphnids	immobilised daphnids (% of introduced)	total number of immobilised daphnids	immobilised daphnids (% of introduced)
[mg test item/L]	x	[24 h]	[24 h]	[48 h]	[48 h]
Control	20	0	0.0	0	0.0
0.81	20	0	0.0	0	0.0
2.28	20	0	0.0	0	0.0
6.38	20	0	0.0	0	0.0
17.9	20	1	5.0	1	5.0
50.0	20	15	75.0	19	95.0

 

Sublethal effects on the mobile daphnids were not observed except for one individual daphnid trapped at the water surface after 48 h at 0.81 mg/L. Full details are given in section

 

Analytical Results (Summary)

The test item concentrations were stable during the test period. The analytical results are summarised in the table below.

Table 6.1.3/2: Summary of measured concentrations during the test.

Nominal concentration [mg test item/L]	test period [d]	age of test solution [d]	measured concentration [mg test item/L]	recovery in % of nominal concentration	geometric mean measured concentration over 48 h [mg test item/L]	recovery in % of nominal concentration over 48 h
0	(control)	0	0	<LOD	-	-
0.81	0	0	0.68	84.0	-	-
2.28	0	0	2.18	95.6	-	-
6.38	0	0	6.39	100	-	-
17.9	0	0	16.5	92.2	-	-
50.0	0	0	47.3	94.6	-	-
0	(control)	2	2	<LOD	n.a.	<LOD
0.81	2	2	0.42	51.9	0.534	65.9
2.28	2	2	1.42	62.3	1.76	77.2
6.38	2	2	3.32	52.0	4.61	72.3
17.9	2	2	9.0	50.3	12.2	68.2
50.0	2	2	24.9	49.8	34.3	68.6

Limit of quantification (LOQ): 0.4 mg/L; < LOD: below the limit of detection (i.e. < 0.12 mg/L); n.a.: not applicable

Since the geometric mean of the measured concentrations was not between 80 and 120% of nominal, the biological endpoints are expressed based on nominal and on measured concentrations.

No significant interferences from the test medium were detected at the retention time corresponding to the analyte in any of the control specimens.

 

The validity criteria are immobilised daphnids in the control should be less than or equal to10% and dissolved oxygen concentration in control and test vessels at the end of the test should be greater than or equal to 3 mg/L. Signs of disease or stress, for example, discoloration or unusual behaviour such as trapping at surface of water, did not exceed 10 per cent of the daphnids in the controls.

The No Observed Effect Concentration (NOEC) based on statistical evaluation of immobility is 17.9 mg/L based on nominal concentrations. This corresponds to a mean measured concentration of 12.2 mg/L. No considerable sub-lethal effects were observed at or below this same test concentration.

Validity criteria fulfilled:

yes

Conclusions:

The 48-hour acute toxicity of test substance to the Daphnia magna, was determined in accordance with the requirements of OECD Chemicals Testing Guideline No. 202 Daphnia sp. Acute Immobilisation Test. Based on geometric mean measured concentrations, the 48-hour EC50 value was determined to be 20.5 mg/L. The No Observed Effect Concentration (NOEC) based on statistical evaluation of immobility is 17.9 mg/L based on nominal concentrations which corresponds to a mean measured concentration of 12.2 mg/L.

Executive summary:

The 48-hour acute toxicity of test substance to the Daphnia magna was conducted in accordance with the requirements of OECD Guideline 202 Daphnia sp. Acute Immobilisation Test.

 

Based on the results of a range-finding test, for which the key results only have been reported, the definitive test was conducted at nominal concentrations of 0.81, 2.28, 6.38, 17.9 and 50.0 mg/L. A control group was also included.

 

At the start of the test, five juvenile Daphnia magna were added to each test vessel. The Daphnia magna in each test vessel were observed at 24 and 48 hours. The Daphnia magna were not fed during the test. Analysis of the test media samples was conducted at 0 (fresh media) and 48 hours (old media). Analysis of the freshly prepared test media at 0 hours showed measured concentrations to range from 66 to 77% of nominal.

 

The No Observed Effect Concentration (NOEC) based on statistical evaluation of immobility is 17.9 mg/L based on nominal concentrations which corresponds to a mean measured concentration of 12.2 mg/L. In terms of geometric mean measured concentrations the 48-hour EC50 value was determined to be 20.5 mg/L. The validity criteria for control immobility (≤10%) and dissolved oxygen (>3 mg/L) were both satisfied.

Description of key information

OECD Guideline 202, GLP, key study, validity 1:

48h-EC50 (Daphnia magna) = 20.5 mg/L based on measured geometric mean concentration

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

20.5 mg/L

Additional information

The 48-hour acute toxicity of test substance to the Daphnia magna was conducted in accordance with the requirements of OECD Guideline 202 Daphnia sp. Acute Immobilisation Test.
 
Based on the results of a range-finding test, for which the key results only have been reported, the definitive test was conducted at nominal concentrations of 0.81, 2.28, 6.38, 17.9 and 50.0 mg/L. A control group was also included.
 
At the start of the test, five juvenile Daphnia magna were added to each test vessel. The Daphnia magna in each test vessel were observed at 24 and 48 hours. The Daphnia magna were not fed during the test. Analysis of the test media samples was conducted at 0 (fresh media) and 48 hours (old media). Analysis of the freshly prepared test media at 0 hours showed measured concentrations to range from 66 to 77% of nominal.
 
The No Observed Effect Concentration (NOEC) based on statistical evaluation of immobility is 17.9 mg/L based on nominal concentrations which corresponds to a mean measured concentration of 12.2 mg/L. In terms of geometric mean measured concentrations the 48-hour EC50 value was determined to be 20.5 mg/L. The validity criteria for control immobility (≤10%) and dissolved oxygen (>3 mg/L) were both satisfied.