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 fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

In the preliminary test, the measured exposure concentrations were well maintained after 24 hours of exposure at the highest tested concentration (10 mg/L). Indeed, 80% of the nominal concentration was maintained after 24 hours exposure interval. However, this stability was not observed in the definitive test. First of all, the initial measured concentrations at each interval time were lower than the nominal values (3 to 4 mg/L for a nominal concentration of 9 mg/L). The samples at the end of the exposure interval after 24 hours were measured after 24 hours storage due to technical problems. After that, the measured concentration for the highest tested concentration (9 mg/L nominal) at the end of the exposure intervals after 72 hours and 96 hours were maintenant at 67% and 128% of the initial concentration. Due to this inconsistency, this study is considered reliable with restrictions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

photoperiod was 17 hours daily. The guideline recommends 12 to 16 hours photoperiod daily. However this is not thought to affect the outcome of the study.

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

yes

Remarks:

photoperiod was 17 hours daily. The guideline recommends 12 to 16 hours photoperiod daily. However this is not thought to affect the outcome of the study.

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: 0 (control), 1.00, 1.73, 3.00, 5.20 and 9.00 mg/l
- Sampling method: Analytical evaluation of various concentrations of the test item and the control was carried via GC-MS from freshly prepared media after 0, 48 and 72 h and from corresponding aged test media after 24, 72 and 96 h.
- Sample storage conditions before analysis: Original samples were stored at room temperature before preparation, if necessary. All prepared samples were stored at room temperature (in autosampler) before start of analysis, if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Directly weighing. Appropriate amounts of the test item were weighed for each test concentration and were transferred into a 10000 mL vessel each. The test medium was stirred for 24 h on a magnetic stirrer at 30 ± 1 °C. Thereafter, the test solutions were allowed to stand at 21 - 23 °C (test temperature) for 24 h.

- Controls: 7 fish in dilution water (without test item) were tested under the same test conditions as the test replicates.

- Evidence of undissolved material (e.g. precipitate, surface film, etc): Not reported

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: Brood stock from DR.NOACK-LABORATORIEN, originally from Umweltbundesamt, Schichauweg 58, D-12307 Berlin
- Age at study initiation (mean and range, SD): Not reported
- Length at study initiation (length definition, mean, range and SD): Average body length at the test start: 2.79 cm
- Weight at study initiation (mean and range, SD): Average body weight at the test start: 0.354 g
- Method of breeding: Not reported
- Feeding during test: None
- Food type: n/a
- Amount: n/a
- Frequency: n/a

ACCLIMATION
- Acclimation period: 12 days
- Acclimation conditions (same as test or not): Yes
- Type and amount of food: 4 % of the fish body weight per feeding day
- Feeding frequency: 3 times per week
- Health during acclimation (any mortality observed): No mortality during acclimation

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

62 mg CaCO3/L

Test temperature:

22.8 - 23.7 °C

pH:

7.05 - 7.76

Dissolved oxygen:

76 - 100 %

Nominal and measured concentrations:

Nominal: 0 (control), 1.00, 1.73, 3.00, 5.20 and 9.00 mg/l.
Measured: 0 (control), 0.258, 0.523, 1.27, 2.26 and 3.84 mg/l (see attached document in "Attached background material")

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria
- Type: covered with a floating lid
- Material, size, headspace, fill volume: Glass, 13 L, filled with 10 L of dilution water (dimensions: 25.5/18128 cm, depth of water: 20 cm).
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): Daily renewal
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 7
- No. of vessels per control (replicates): 1
- Biomass loading rate: Fish density in the test vessels was 0.248 g fish per litre test solution. Average body length at the test start: 2.79 cm. Average body weight at the test start: 0.354 g.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water of local origin was used for holding and testing. The water was filtered on activated charcoal and aerated for at least 24 hours to remove chlorine. Nominal water parameters:
Hardness: 10 - 250 mg CaC03/L
pH-value: 6.0 - 8.5
- Culture medium different from test medium: No
- Intervals of water quality measurement: pH-value. Temperature and oxygen saturation were measured in all test vessels at the beginning and at the end of the exposure and every 24 hours from fresh and old test media. Total hardness of the water was determined at the beginning out of the control. During the test the water temperature was recorded continuously with a data logger. The light intensity on the surface of the test aquaria was measured at test start.
Analytical evaluation of various concentrations of the test item and the control was carried via GC-MS from freshly prepared media after 0, 48 and 72 h and from corresponding aged test media after 24, 72 and 96 h.

OTHER TEST CONDITIONS
- Adjustment of pH: None reported
- Photoperiod: A daily 17 hour photoperiod was maintained throughout exposure.
- Light intensity: 0.1 - 10 µmol photons· m-2 S-1 (corresponding to approximately 7 - 750 Lux).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Death or visible abnormalities (e.g. loss of equilibrium, swimming behaviour, respiratory function, pigmentation, etc.) after 2-4, 24, 48, 72 and 96 h.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.73

- Range finding study
- Test concentrations:
Nominal values: 0, 0.1, 1.0, 10 mg/L
Measured values at the start of the exposure interval (0h): < SysQL, 0.104, 1.08, 9.10 mg/L
Measured values at the end of the exposure interval (24h): < SysQL, 0.0663, 0.654, 8.03 mg/L
- Results used to determine the conditions for the definitive study: Observations on mortality and abnormal behaviour at each concentration.
33% mortality at 96 h was observed in the 0.1 mg/l concentration, 0% mortality in the 1.0 mg/l concentrations and 100% mortality by 24 h was observed in the 10 mg/l concentration.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

3.769 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL 3.763 - 3.774

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

8.82 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL 8.80 - 8.83

Details on results:

- Behavioural abnormalities: See Table 1
- Observations on body length and weight: Not reported
- Other biological observations: See Table 1
- Mortality of control: None
- Other adverse effects control: No
- Abnormal responses: See Table 1
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None observed
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

LC10.20.50-values at 48 h, 72 hand 96 h were calculated by sigmoidal dose-response regression. No LC-values were calculated for 2 and 24 h since no mortality occurred. The geometric mean measured values after 96 hours were used for all corresponding calculations. The concentrations leading to 0 and 100 % mortality (LCo and LC100) after 96 h were determined directly from the test results. The chosen methods for determination of LCx-values were in accordance with the OECD series on testing and assessment No. 54 (2006).
The data for the tables in this report were computer generated and have been rounded for presentation from the full derived data. Consequently if calculated manually based on the given data minor variations may occur from these figures. The following software was used for calculations:
Excel, MICROSOFT CORPORATION
GraphPad Prism, GRAPHPAD SOFTWARE, INC.

Sublethal observations / clinical signs:

Table 1 Observations in the Definitive Test (7 fish per concentration and control) 

Nominal Test Item Concentration (mg/l)	Observation	Number of fish affected after different exposure periods [hours]
		2	24	48	72	96
9	(E)	-	-	2	2	1
	(4.1)	-	7	5	3	2
	(2.3)	-	-	1	-	-
	(2.1)	7	7	4	3	2
5.20	(4.1)	7	7	7	7	7
	(2.5)	7	5	5	5	5
	(2.1)	-	2	2	2	2
3.0	(2.4)	-	7	7	7	-
	(1)	7	-	-	-	7
1.73	(1)	7	7	7	7	7
1	(1)	7	7	7	7	7
Control	(1)	7	7	7	7	7

*) The numbers in brackets correspond to the following observations:

(1) = Normal behaviour

(2.1) = Lethargic

(2.3) = Staggering

(2.4) = Slowed escape reflex

(2.5) = Missing escape reflex

(4.1) = Hyperventilation

(E) = Exitus lethalis

Table 2 Cumulative Mortality [%] in the Definitive Test (7 fish per concentration and control)

Nominal test item concentration [mg/L]	Cumulative Mortality [%] after different exposure periods [hours]
	2	24	48	72	96
9.0	0	0	29	57	71
5.2	0	0	0	0	0
3.0	0	0	0	0	0
1.73	0	0	0	0	0
1	0	0	0	0	0
Control	0	0	0	0	0

Validity criteria fulfilled:

yes

Conclusions:

A 96 hr LC50 value of 3.769 mg/l (measured geometric mean) was determined in a reliable study of short-term toxicity to fish, conducted according to an appropriate test protocol, and in compliance with GLP.

Executive summary:

The acute toxicity of the test substance to fish (zebrafish) was determined according to the OECD Test Guideline 203 and EU Method C.1 with GLP statement. A semi-static test with daily renewal of the test media was conducted with the nomial test item concentrations of 1.00, 1.73, 3.00, 5.20 and 9.00 mg/L (factor 1.73) corresponding to geometric mean measured concentrations of 0.258, 0.523, 1.27, 2.26 and 3.84 mg/L. Duration of the test was 96 hours. 7 test organisms were exposed to each test concentration and the control. Water quality parameters temperature, pH-value and oxygen-saturation measured at 0, 24, 48, 72 and 96 hours were determined to be within the acceptable limits. The determination of the test concentrations of the test substance was carried out via GC-MS. Evaluation was carried out from freshly prepared media after 0, 48 and 72h and from the corresponding 24h aged test media after 24, 72 and 96h. The measured concentration of the test substance in freshly prepared media after 0, 48 and 72h of exposure were in the range of 23 to 91% of the nominal values. The measured concentrations in the corresponding 24h aged test media were in the range of <LOQ to 63% of the nominal values. Based on this, the effect values are based on geometric mean measured test substance concentrations. Additionally, nominal test substance concentrations are given. Evaluation for all time points was done with geometric mean measured values after 96 hours. All validity criteria of the test guideline were met. the 96 h LC₅₀ for Danio rerio was determined at 3.769 mg/l (95% confidence interval: 3.763 – 3.774 mg/L.

Description of key information

EU Method C.1, OECD Guideline 203, GLP, key study, validity 2:
96h-LC50 (Danio rerio) = 3.769 mg/L (95% CI: 3.763 -3.774 mg/L) based on geometric mean measured concentrations

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

3.769 mg/L

Additional information

One key study is available to assess the 96h acute toxicity of the registered substance to zebra fish (Danio rerio) under semi-static conditions with a daily test medium renewal according to the EU Method C.1 and the OECD Guideline 203, with GLP statement.

The nominal test substance concentrations tested were 1.00, 1.73, 3.00, 5.20 and 9.00 mg/L corresponding to geometric mean measured concentrations of 0.258, 0.523, 1.27, 2.26 and 3.84 mg/L. The measured concentration of the test substance in freshly prepared media after 0, 48 and 72h of exposure were in the range of 23 to 91% of the nominal values. The measured concentrations in the corresponding 24h aged test media were in the range of <LOQ to 63% of the nominal values. Based on this, the effect values are based on geometric mean measured test substance concentrations.

All validity criteria were fulfilled. The 96h-LC50 value was determined at 3.769 mg/L (95% confidence interval: 3.763 – 3.774 mg/L; geometric mean measured concentrations).