1,3,3-trimethylnorbornan-2-one

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:

09 February 2018 – 07 July 2018

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:

yes

Remarks:

Some analysed concentrations of test item were outside the acceptable limit. This deviation does not have any impact on the study since test item was found stable in test medium and the derived EC50 is far from LOQ of analytical method.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (control), 0.3, 0.66, 1.45, 3.19, 7.02, 15.44 and 34 mg/L
- Sampling method: All the test concentrations along with the negative control were analyzed for the test item concentration at the beginning and end of test. Test item stock (100 mg/L) was analysed at the start of test. For analysis, single composite sample was drawn from prepared test concentrations and duplicate sample was drawn from the negative control.
- Sample storage conditions before analysis: not applicable

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test item stock of 0.1 mg/mL (100 mg/L) was prepared by mixing 100 mg test item and making up to 1 L in a volumetric flask using test medium. To prepare test concentrations of 0.3, 0.66, 1.45, 3.19, 7.02, 15.44 and 34 mg/L, 0.3, 0.66, 1.45, 3.19, 7.02, 15.44 and 34 mL of test item stock was added and made up to 1 L in a volumetric flask using test medium separately.
- Negative control: 1 L test medium was used.
- Positive control: A stock solution of reference substance (Potassium dichromate- K2Cr2O7) at 1 mg/mL was prepared by mixing 10 mg in 10 mL of test medium. For preparation of reference substance at 0.32 mg/L, 0.32 mL of reference substance stock was mixed and made up to 1 L in a volumetric flask using test medium.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Source: Ecotoxicology, Eurofins Advinus Limited, Bengaluru, INDIA.
- Age of parental stock (mean and range, SD): no data
- Feeding during test: No

ACCLIMATION
- Acclimation period: 24 h

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

None

Hardness:

237 mg/L as CaCO3

Test temperature:

20.2 to 20.3ºC

pH:

7.79 to 7.82

Dissolved oxygen:

7.0 to 7.3 mg O2/L

Salinity:

81 mg/L as CaCO3 (alkalinity)

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 0.3, 0.66, 1.45, 3.19, 7.02, 15.44 and 34 mg/L.
Mean measured concentrations (after 48 h): 0 (control), ND, ND, 1.159, 3.152, 6.432, 14.31 and 31.88 mg/L.
Nominal concentrations were considered to calculate the EC50 value.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beaker
- Material, size, headspace, fill volume: glass, 250 mL capacity, 150 mL fill volume
- Aeration: The test containers were not bubble aerated.
- Renewal rate of test solution (frequency/flow rate): Not applicable (static test)
- 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): Not applicable
- Biomass loading rate: 30 mL of test media per daphnia.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water (ISO test water)
- Intervals of water quality measurement: pH and dissolved oxygen concentration of test concentrations at the start and end of the treatment were recorded. The temperature of the incubator/growth chamber was recorded once daily during the test.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: complete darkness

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : All test daphnids were observed for immobility at 24, and 48 h of exposure.

VEHICLE CONTROL PERFORMED: Not applicable
RANGE-FINDING STUDY
- Test concentrations: 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100 mg test item/L along with negative control.
- Results used to determine the conditions for the definitive study: In the range finding test the per cent immobilisation of daphnia was 0, 0, 0, 0, 0, 40, 90 and 100% at the tested concentrations of 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100 mg/L, respectively. Based on these results, the test item concentrations of 0.3 (G2), 0.66 (G3), 1.45 (G4), 3.19 (G5), 7.02 (G6), 15.44 (G7) and 34 (G8) mg/L (factor of 2.2) was selected for the definitive test along with negative control and reference substance.

Reference substance (positive control):

yes

Remarks:

(potassium dichromate)

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

9.744 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: fiducial limit at 95 % was 4.4998 to 31.0071 mg/L

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

4.373 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: fiducial limit at 95 % was 3.2383 to 5.9043 mg/L

Details on results:

- Behavioural abnormalities: Not specified.
- Mortality of control: No mortality was observed over a period of 48 h in the control group.
- Other: A definitive test was performed however the analysis of prepared test concentrations indicated that the mean per cent agreement was varied between 0 and 30% with the claimed concentrations. Hence, definitive test-II was carried out with the same test concentrations.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: At 0.32 mg/L reference substance, the immobilization of daphnia was 35 and 60 % observed at 24 and 48 hours exposure, respectively.
- EC50-48 h: < 0.32 mg/L

Reported statistics and error estimates:

The EC50 value with 95% fiducial limits, and the equation for the dose-response curve was determined by Probit analysis by the method of Finney (1971) using developed and validated computer programme.

Table 1: Dissolved Oxygen Concentration and pH of Test Concentrations

Group	Treatment (mg/L)	Dissolved oxygen concentration (mg(L)		pH
		Start of test	End of start  (48 hours)	Start of test	End of start  (48 hours)
G1	Negative control	6.5	6.4	7.79	7.80
G2	0.3	6.8	6.6	7.78	7.79
G3	0.66	6.6	6.5	7.81	7.82
G4	1.45	6.7	6.5	7.79	7.80
G5	3.19	6.7	6.6	7.80	7.81
G6	7.02	6.6	6.5	7.81	7.82
G7	15.44	6.6	6.5	7.79	7.80
G8	34	6.6	6.5	7.78	7.79
G9	Reference substance 0.32	6.5	6.4	7.79	7.81

Table 2: Results on Daphnia Immobilization

Group	Treatment (mg/L)	No. of Daphnia immobilized (5 Daphnia per replicate)								% Immobilization
		24 h				48 h				24 h	48 h
		R1	R2	R3	R4	R1	R2	R3	R4
G1	Negative control	0	0	0	0	0	0	0	0	0	0
G2	0.3	0	0	0	0	0	0	0	0	0	0
G3	0.66	0	0	1	0	1	1	1	0	5	15
G4	1.45	0	0	1	0	1	0	2	0	5	15
G5	3.19	1	0	0	1	2	2	1	2	10	35
G6	7.02	1	1	0	1	2	3	2	3	15	50
G7	15.44	4	4	5	5	4	5	5	5	90	95
G8	34	3	5	5	4	5	5	5	5	85	100
G9	Reference substance 0.32	2	1	2	2	3	3	2	4	35	60

Table 3: Results of Test Concentration Analysis at the End of Test

Claimed Con  (mg/ L)	Sample Code	Analyzed Test Item Conc        (mg/L)	% Agreement with Claimed Conc.	Final Test Item Conc.			Overall % Agreement with Claimed Conc.
				Mean	SD	%RSD	Mean	SD	%RSD
0.30	G2R1-1	0	0	0	0	0	0	0	0
	G2R1-2	0	0
	G2R1-3	0	0
0.66	G3R1-1	0	0	0	0	0	0	0	0
	G3R1-2	0	0
	G3R1-3	0	0
1.45	G4R1-1	1.044	71.99	1.159	0.100	8.6	79.92	6.87	8.6
	G4R1-2	1.216	83.87
	G4R1-3	1.217	83.90
3.19	G5R1-1	3.169	99.33	3.152	0.172	5.5	98.81	5.39	5.5
	G5R1-2	3.315	103.9
	G5R1-3	2.972	93.17
7.02	G6R1-1	6.000	85.47	6.432	0.408	6.3	91.62	5.81	6.3
	G6R1-2	6.811	97.03
	G6R1-3	6.484	92.37
15.44	G7R1-1	13.36	86.52	14.31	1.05	7.3	92.67	6.81	7.3
	G7R1-2	15.44	99.99
	G7R1-3	14.13	91.51
34.00	G8R1-1	33.12	97.41	31.88	1.12	3.5	93.77	3.29	3.5
	G8R1-2	31.58	92.90
	G8R1-3	30.94	91.01

Conclusion: There was no interference in the G1 samples at the RT of the analyte. The obtained results of G5, G6, G7 and G8 were within the acceptance limits (80-120%).

Validity criteria fulfilled:

yes

Remarks:

(mortality in control < 10% (no immobilisation observed); dissolved oxygen concentration > 3 mg O2/L)

Conclusions:

In a short-term toxicity test to daphnia magna, the 48h-EC50 of the test substance was found to be 4.373 mg/L.

Executive summary:

An acute aquatic toxicity study with Daphnia magna was conducted on test item according to OECD guideline 202, following GLP. In a preliminary range finding study the per cent immobilisation of daphnia was 0, 0, 0, 0, 0, 40, 90 and 100% at the tested concentrations of 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100 mg/L, respectively. Based on these results, the definitive test was conducted with test item concentrations of 0.3, 0.66, 1.45, 3.19, 7.02, 15.44 and 34 mg/L (factor of 2.2) for a period of 48 h in static conditions in 4 replicates of 5 daphnids per replicate. Reconstituted water (ISO test water) was used as test medium. No vehicle was used since the test item was found soluble in test medium at the highest concentration tested. Negative control consisted of test medium alone and a solution of potassium dichromate was used as reference substance. A validated analytical method based on HPLC was used to monitor the concentration and stability of the active ingredient in the test solution. The analysis of test concentrations 1.45, 3.19, 7.02, 15.44 and 34 mg/L showed an agreement with claimed concentrations of 79.92 to 98.81 % at end of test (48 hour). Test concentrations of 0.3, 0.66, 1.45 and 7.02 mg/L were outside the acceptable limit (80-120% of nominal). This deviation does not have any impact on the study since stability test found that test item is stable in test medium between 1.06 and 106 mg/L and the derived EC50 is far from LOQ of analytical method. Thus, nominal concentrations were considered to calculate the EC50 value. There was no immobilization of daphnia in the negative control and dissolved oxygen was higher than 3 mg/L in control and test vessels at the end of the test. The immobilization of daphnia was 0, 15, 15, 35, 50, 95 and 100 % at 48 hours exposure at 0.3, 0.66, 1.45, 3.19, 7.02, 15.44 and 34 mg/L. Based on these results, the 48h-EC50 value was 4.373 mg/L with 3.2383 to 5.9043 mg/L confidence limits at 95 %.

Description of key information

Key study. Test method according to OECD 202, GLP study. The 48h-EC50 of the test substance to Daphnia magna was found to be 4.373 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

4.373 mg/L

Additional information

Key study: An acute aquatic toxicity study with Daphnia magna was conducted on test item according to OECD guideline 202, following GLP. In a preliminary range finding study the per cent immobilisation of daphnia was 0, 0, 0, 0, 0, 40, 90 and 100% at the tested concentrations of 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100 mg/L, respectively. Based on these results, the definitive test was conducted with test item concentrations of 0.3, 0.66, 1.45, 3.19, 7.02, 15.44 and 34 mg/L (factor of 2.2) for a period of 48 h in static conditions in 4 replicates of 5 daphnids per replicate. Reconstituted water (ISO test water) was used as test medium. No vehicle was used since the test item was found soluble in test medium at the highest concentration tested. Negative control consisted of test medium alone and a solution of potassium dichromate was used as reference substance. A validated analytical method based on HPLC was used to monitor the concentration and stability of the active ingredient in the test solution. The analysis of test concentrations 1.45, 3.19, 7.02, 15.44 and 34 mg/L showed an agreement with claimed concentrations of 79.92 to 98.81 % at end of test (48 hour). Test concentrations of 0.3, 0.66, 1.45 and 7.02 mg/L were outside the acceptable limit (80-120% of nominal). This deviation does not have any impact on the study since stability test found that test item is stable in test medium between 1.06 and 106 mg/L and the derived EC50 is far from LOQ of analytical method. Thus, nominal concentrations were considered to calculate the EC50 value. There was no immobilization of daphnia in the negative control and dissolved oxygen was higher than 3 mg/L in control and test vessels at the end of the test. The immobilization of daphnia was 0, 15, 15, 35, 50, 95 and 100 % at 48 hours exposure at 0.3, 0.66, 1.45, 3.19, 7.02, 15.44 and 34 mg/L. Based on these results, the 48h-EC50 value was 4.373 mg/L with 3.2383 to 5.9043 mg/L confidence limits at 95 %.