Reaction mass of 3-(3,3-dimethyl-2,3-dihydro-1H-inden-5-yl)propanal and 3-(1,1-dimethyl-2,3-dihydro-1H-inden-5-yl)propanal and 3-(1,1-dimethyl-2,3-dihydro-1H-inden-4-yl)propanal

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:

17th to 21st November 2014

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

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 2013-06-03/05. Date of signature: 2013-11-05

Analytical monitoring:

yes

Details on sampling:

- Concentrations: controls and all treatment levels
- Sampling method: All concentration levels and the control were analytically verified the test item via GC-MS at the start (0 and 24 h) and at the end of both exposure intervals (24 and 48 h).
- Sample storage conditions before analysis: Samples were analysed at the day of collection. For possible re-analyses, samples were stored in tightly closed vials without headspace at 6 ± 2 °C. Re-analyses were not necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solutions (9.00 mg/L of the test item were weighed out) were prepared with dilution water two days before the start of the exposure intervals (at -48 and -24h). The stock solutions were prepared with dilution water and preincubated at 30 ± 2 °C with constant stirring with approximately 1100 rpm for 24 h. Thereafter the stock solutions were allowed to stand at 20 ± 1 cC (test temperature) for 24 hours. Before preparing the test concentrations the stock solutions were checked for undissolved test item.
Per replicate, 20 g test solution were weighed out into the test vessel. This corresponds to 20 mL per test vessel. The daphnids were inserted with a small amount of dilution water (start of the exposure) or test solution (water renewal) by a pipette.

- Evidence of undissolved material (e.g. precipitate, surface film, etc): Presence of undissolved test item during preparation of the test concentrations and during the test was not observed.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Source: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu),
D-14195 Berlin
- Age at study initiation (mean and range, SD): 2-24 h
- Feeding during test: none

ACCLIMATION
- Acclimation period: At least 2 h in dilution water
- Acclimation conditions (same as test or not): see above
- Type and amount of food: The culture daphnids were fed with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 106 cells/mL. The algae are cultured at the test facility.
- Feeding frequency: at least 5 times per week ad libitum
- Health during acclimation (any mortality observed): not reported

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

263-265 mg CaCO3/L

Test temperature:

20.8-21 °C

pH:

7.6

Dissolved oxygen:

8.75-8.81 mg/L

Nominal and measured concentrations:

nominal: 1.00 - 1.73 - 3.00 - 5.20 - 9.00 mg/L.
geometric mean measured: 0.877 – 1.43 – 2.84 – 4.47 – 7.83 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers (4 cm ID x 7 cm H), 50 mL capacity, loosely covered with watch glasses
- Type: see above
- Material, size, headspace, fill volume: see above, filled with 20 mL volume
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): 24 h - the freshly prepared test solutions were filled into a second set of test vessels and the daphnids were transferred by a pipette
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO Test water, according to OECD 202, Annex 3
- Conductivity: 650-667 µS/cm
- Culture medium different from test medium: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
- Intervals of water quality measurement: Prior to the start of the exposure (0 h) and the water renewal (24 h), the water quality parameters (i.e. pH value, dissolved oxygen concentration, temperature, conductivity and total hardness) of the dilution water were measured.

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: 16/8 h light/dark cycle
- Light intensity: Diffuse light; light intensity of max. 20 µE m-2 s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Immobilisation after 24 and 48 hours.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: factor of √3
- Justification for using less concentrations than requested by guideline: n/a
- Range finding study
- Test concentrations: 1, 10, 100 mg/l
- Results used to determine the conditions for the definitive study: 100% immobilisation at 10 mg/l, 0% immobilisation at 1 mg/l

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

4.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(analytically confimed concentrations)

Basis for effect:

mobility

Remarks on result:

other: Cl: 3.65 – 4.71.

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

6.23 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

(analytically confimed concentrations)

Basis for effect:

mobility

Remarks on result:

other: Cl: 5.44 – 7.30

Details on results:

- Behavioural abnormalities: none reported
- Observations on body length and weight: not analysed
- Other biological observations: none reported
- Mortality of control: 0%
- Other adverse effects control: none reported
- Abnormal responses: none reported
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50/LC50: EC50: 1.57 mg/L (CI 1.53 - 1.60 mg/L)

Reported statistics and error estimates:

The EC50-values after 24 and 48 h were calculated by sigmoidal dose-response regression. The 95 % confidence limits were calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism5.

Table 1:      Immobilisation Ratesafter 24 and 48 h ofExposure in the Definitive Test

                (n = 20, divided into 4 replicates with 5 daphnids each)

Nominal test item concentration   [mg/L]	Geometric mean measured test item concentration [mg/L]	IMMOBILISATION [%]
		24 h					48 h
		Replicates					Replicates
		1.	2.	3.	4.	MV	1.	2.	3.	4.	MV
9.00	7.83	80	80	80	100	85	100	100	100	100	100
5.20	4.47	80	20	20	0	30	100	80	60	80	80
3.00	2.84	0	0	0	0	0	40	0	0	0	10
1.73	1.43	0	0	0	0	0	0	0	0	20	5
1.00	0.877	0	0	0	0	0	0	0	0	0	0
Control		0	0	0	0	0	0	0	0	0	0

MV = mean value

Table 2:      Measured Concentrations of the Test Item during the Definitive Test

Sampling date	2014-11-19 0 hours Start of the exposure interval		2014-11-20 24 hours End of the exposure interval		2014-11-20 24 hours Start of the exposure interval		2014-11-21 48 hours End of the exposure interval
Nominal test item concentration [mg/L]	Reaction Mass of 3-(1,1-dimethyl-4-indanyl)propanal and 3-(1,1-dimethyl-5-indanyl)propanal and 3-(3,3-dimethyl-5-indanyl)propanal								Geometric mean
	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%
9.00	8.36	93	7.39	82	10.6	118	5.75	64	7.83	87
5.20	5.09	98	4.65	89	4.94	95	3.42	66	4.47	86
3.00	3.26	109	2.95	98	3.49	116	1.94	65	2.84	95
1.73	1.62	94	1.28	74	1.82	105	1.11	64	1.43	83
1.00	0.971	97	0.901	90	1.10	110	0.614	61	0.877	88
Control	< LOQ		< LOQ		< LOQ		< LOQ		< LOQ

Meas. conc. = measured concentration of the test item, single injection, dilution factors taken into account

%                  = percent of the nominal concentration of the test item

LOQ             = limit of quantification (0.1 mg test item/L)

Validity criteria fulfilled:

yes

Conclusions:

A 48-hour EC50 value of 4.25 mg/L (nominal, supported by analysis) was determined in a reliable study of short-term toxicity to Daphnia magna, conducted according to an appropriate test protocol, and in compliance with GLP.

Executive summary:

This study was performed according to EU Method C.2 and OECD Guideline 202 with GLP compliance to assess the short-term toxicity of the test substance to aquatic invertebrates. The study was conducted under semi-static conditions over a period of 48 hours with 5 concentrations of the test substance in the range of 1 to 9 mg/L, prepared with dilution water in a geometric series with a separation factor of 3. The stock solution of 9 mg/L was prepared with dilution water two days before the start of the exposure intervals (at 48 and 24h). Each stock solution was pre-incubated at 30 +/- 2°C with constant stirring with approximately 1100 rpm for 24 hours. Thereafter the stock solutions were allowed to stand at 20 +/- 1°C (test temperature) for 24 hours. Further test concentrations were prepared by diluting the stock solution of 9 mg/L with dilution water. The tested solutions were clear throughout the exposure period. Presence of undissolved test item during preparation of the test concentrations and during the test was not observed. Twenty daphnids were exposed to each concentration level and the control. The concentrations of the test substance were analytically verified via GC-MS at the start of the exposure intervals (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours) in all concentration levels and the control. The measured concentrations of the test substance at the start of the exposure intervals were in the range of 93 to 118% of the nominal values. At the end of the exposure intervals, the measured concentrations of the test substance were in the range of 61 to 98% of the nominal values. The geometric mean measured concentrations of the test substance were calculated to be 0.877 - 1.43 - 2.84 - 4.47 - 7.83 mg/L. Since the geometric mean measured concentration for each test concentration is within +/-20% of the respective nominal test substance concentration, the results can be based on the nominal concentrations. The 48 hour EC₅₀for Daphnia magna was determined at 4.25 mg/l (95% confidence interval: 3.65 - 4.71 mg/l). 

The water quality parameters, measured at the start and at the end of the exposure intervals were within the acceptable limits. The validity criteria of the test guidelines were fulfilled.

Description of key information

EU Method C.2, OECD Guideline 202, GLP, key study, validity 1:

48h-EC50 (Daphnia magna) = 4.25 mg/L (95% CI: 3.65 -4.71 mg/L) based on analytically confirmed nominal concentrations

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

4.25 mg/L

Additional information

One key study is available to assess the 48h acute toxicity of the registered substance to Daphnia magna, under semi-static conditions, according to the EU Method C.2 and the OECD Guideline 202 with GLP statement.

The nominal test substance concentrations tested were 1.0, 1.73, 3.0, 5.20 and 9.0 mg/L. The tested solutions were clear throughout the exposure period. Presence of undissolved test item during preparation of the test concentrations and during the test was not observed. Twenty daphnids were exposed to each concentration level and the control. The concentrations of the test substance were analytically verified via GC-MS at the start of the exposure intervals (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours) in all concentration levels and the control. The measured concentrations of the test substance at the start of the exposure intervals were in the range of 93 to 118% of the nominal values. At the end of the exposure intervals, the measured concentrations of the test substance were in the range of 61 to 98% of the nominal values. The geometric mean measured concentrations of the test substance were calculated to be 0.877 - 1.43 - 2.84 - 4.47 - 7.83 mg/L. Since the geometric mean measured concentration for each test concentration is within +/-20% of the respective nominal test substance concentration, the results can be based on the nominal concentrations. The 48 hour EC₅₀for Daphnia magna was determined at 4.25 mg/L (95% confidence interval: 3.65 - 4.71 mg/L).