1,4-Dioxaspiro[4.5]decane, 8-[4-(4-methylphenyl)-3-cyclohexen-1-yl]-

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

Study period:

2018-05-04 to 2018-08-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

Council Regulation (EC) No. 440/2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and the council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 100 mg/L
- Sampling method: samples were taken at the beginning of the exposure period and then daily at 0-h, 24-h, 48-h, 72-h and 96-h for analysis. The samples were used undiluted due to a low solubility of the test item (around LOD of 0.003 mg/L).
- Sample storage conditions before analysis: no,

Vehicle:

yes

Remarks:

Reconstituted water

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The test medium (reconstituted water and test item) was prepared freshly. Therefore, the calibrated flask with test medium was treated in an ultrasonic device for 1 hour. Subsequently, the preparation was stirred with a magnetic stirrer for further 23 hours. Afterwards, the formulation was passed through a membrane filter with a pore size of 0.2 µm. The filtrate was used for the study.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Strain: Danio rerio (Tübinger strain)
- Source: Max-Planck-Institute, Tübingen, Germany
- Length at study initiation: Length per group 2.3 – 2.9 cm (control group) and 2.4 – 2.9 cm (test item group)
- Weight at study initiation: Body weight per group: 0.19 – 0.38 g (control group) and 0.24 – 0.38 g (test item group)
- Feeding during test: commercial fish diet during acclimatization

ACCLIMATION
- Acclimation period: Before start of exposure, the fish were kept in a glass aquarium for at least 12 days in an airconditioned laboratory at a water temperature of 21 - 25°C and a 12 hour light and 12 hour dark regime to ensure similar conditions as in the experiment. Aerated reconstituted water was used as holding medium.
- Acclimation conditions: same as test
- Type and amount of food during acclimation: The fish were fed with Tetra Min® diet and frozen artemia.
- Feeding frequency during acclimation: every day
- Health during acclimation (any mortality observed): No

FEEDING DURING TEST: No

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

about 251.7 mg CaCO3/L

Test temperature:

20.6 - 22.4 °C

pH:

7.56 - 7.92

Dissolved oxygen:

73.5 - 97.1%

Conductivity:

676 µS/cm

Nominal and measured concentrations:

Nominal concentration: 100 mg/L;
Measured concentration: < LOD

Details on test conditions:

TEST SYSTEM
- Test vessel: 10 L glass aquariums
- Type: open
- Aeration: no
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water according to OECD 203
- Ca/mg ratio: 4:1
- Culture medium different from test medium: no
- Intervals of water quality measurement: daily

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 12 hour light and 12 hour dark regime

EFFECT PARAMETERS MEASURED: mortality and clinical symptoms

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: > 0.003 mg/L (LOD)

Details on results:

The 96 hour LC50 for the test material to Zebrafish could not be quantified due to the absence of toxicity at the highest test concentration achievable in water. An aqueous filtrate of 0.1 g/L nominally revealed no aquatic toxicity in the test system.


The 96h LC50 exceeded the LOD of 0.003 mg/L (nominal >100 mg/L) and, thus, could not be determined in this study.


- Behavioural abnormalities: no
- Observations on body length and weight: no
- Other biological observations: no
- Mortality of control: no
- Other adverse effects control: no
- Abnormal responses: no
- 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: yes

Sublethal observations / clinical signs:

Validity of the Test

No mortality was seen during the acclimation phase. Therefore, the acceptance criteria for the fish batch were fulfilled.

The mortality in the control did not exceed 10% at the end of the study.

Constant conditions were maintained throughout the study.

The dissolved oxygen concentration was > 60% throughout the study.

Thus, the validity criteria were fulfilled.

 

Biological Results

Mortality: No mortality occurred.

Clinical Symptoms: No clinical symptoms were observed.

 

LC₅₀-Values

 

Test Duration	LC50
24h	>100 mg/L
48h	>100 mg/L
72h	>100 mg/L
96h	>100 mg/L

 

Analytical Results

Nominal and corresponding analytical concentrations of the control and test medium samples are given in the following table:

 

 

Nominal Test Item Conc. [mg/L]	0 hours		24 hours		48 hours		72 hours		96 hours*
	Meas. Conc. [mg/L]	[%]	Meas. Conc. [mg/L]	[%]	Meas. Conc. [mg/L]	[%]	Meas. Conc. [mg/L]	[%]	Meas. Conc. [mg/L]	[%]
0	<LOD	-	<LOD	-	<LOD	-	<LOD	-	<LOD	-
100.0	<LOQ	-	<LOQ	-	<LOD	-	<LOQ	-	<LOQ	-

LOD = Limit of detection (0.003 mg/L)

LOQ = Limit of quantification (0.01 mg/L)

* Repeat measurement on day 03.06.2018 of the 96 hours samples

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study, an aqueous solution of 100 mg/L of the test item revealed no aquatic toxicity in the test system. The 96h LC50 was > 100 mg/L and, thus, could not be determined in this study.

Executive summary:

The acute toxicity of the test item to zebrafish Danio rerio was determined in a study according to OECD TG 203 and EU method C.1. For this purpose, 10 zebrafish per test group and control were exposed over a 96-h period under defined conditions. The study comprised of one control group and one test item group. A nominal test item concentration of 100 mg/L was prepared in reconstituted water. The zebrafish were observed for signs of toxicity and mortality for 96 hours. The analytical determination of the test item concentration was performed using HPLC with UV-detection. Samples were collected at the beginning of the test (0-h) and daily until the end of the exposure period (96-h). The limit of quantification (LOQ) of the analytical method was 0.01 mg/L. The measured concentrations of the control media were below LOD (0.003 mg/L) at all measuring time points while the measured concentrations of the test media with a nominal concentration of 100 mg/L were below LOD (0.003 mg/L) at 48 hours and below LOQ (0.01 mg/L) at 0, 24, 72 and 96 hours. The tested concentration could not be quantified. Therefore, the LC₅₀  is based on the nominal concentration.

For the test material, the following LC₅₀  values for zebrafish were determined:

24 h LC₅₀ > 0.003 mg/L (nominal > 100 mg/L)

48 h LC₅₀ > 0.003 mg/L (nominal > 100 mg/L)

72 h LC₅₀ > 0.003 mg/L (nominal > 100 mg/L)

96 h LC₅₀ > 0.003 mg/L (nominal > 100 mg/L)

No acute toxicity was observed at a nominal concentration of 100 mg/L, i.e. up to the maximum water solubility. The 96h LC₅₀ was > 100 mg/L (nominal) and, thus, could not be determined in this study.

Description of key information

Under the conditions of the present study, an aqueous solution of 100 mg/L of the test item revealed no aquatic toxicity in the test system. The 96h LC50 was > 100 mg/L and, thus, could not be determined in this study (reference 6.1.1-1).

Additional information

The acute toxicity of the test item to zebrafish Danio rerio was determined in a study according to OECD TG 203 and EU method C.1. For this purpose, 10 zebrafish per test group and control were exposed over a 96-h period under defined conditions. The study comprised of one control group and one test item group. A nominal test item concentration of 100 mg/L was prepared in reconstituted water. The zebrafish were observed for signs of toxicity and mortality for 96 hours. The analytical determination of the test item concentration was performed using HPLC with UV-detection. Samples were collected at the beginning of the test (0-h) and daily until the end of the exposure period (96-h). The limit of quantification (LOQ) of the analytical method was 0.01 mg/L. The measured concentrations of the control media were below LOD (0.003 mg/L) at all measuring time points. The measured concentrations of the test media with a nominal concentration of 100 mg/L were below LOD (0.003 mg/L) at 48 hours and below LOQ (0.01 mg/L) at 0, 24, 72 and 96 hours. The tested concentration could not be quantified. Therefore, the LC₅₀  is based on the nominal concentration.

For the test material, the following LC₅₀  values for zebrafish were determined:

24 h LC₅₀ > 0.003 mg/L (nominal > 100 mg/L)

48 h LC₅₀ > 0.003 mg/L (nominal > 100 mg/L)

72 h LC₅₀ > 0.003 mg/L (nominal > 100 mg/L)

96 h LC₅₀ > 0.003 mg/L (nominal > 100 mg/L)

No acute toxicity was observed at a nominal concentration of 100 mg/L, i.e. up to the maximum water solubility. The 96h LC₅₀ was > 100 mg/L (nominal) and, thus, could not be determined in this study.