Indene

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 Aug 2018 - 26 Sep 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Indene

EC Number:

202-393-6

EC Name:

Indene

Cas Number:

95-13-6

Molecular formula:

C9H8

IUPAC Name:

indene

Test material form:

liquid

Details on test material:

Appereance: Colorless transparent liquid
Storage conditions: At room temperature container flushed with nitrogen

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All test concentrations and the control
- Sampling method: 2.0 mL from the approximate centre of the test vessels at t=0 h and t=48 h
- Sample storage conditions before analysis: The test samples were diluted in a 1:1 (v:v) ratio with acetonitrile and stored in the freezer(≤ -15°C) until analysis at the analytical laboratory of the Test Facility.
- At the end of the exposure period, the replicates were not pooled at each concentration before sampling due to the volatility of the test item. Instead, samples were taken from one individual replicate per treatment group.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- The batch of the test item was a colorless transparent liquid with a purity of 96.9% which was not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test item. All glassware used during the preparation of test solutions was closed with minimal headspace in order to minimize vaporization of the test item.
- Method: Preparation of test solutions started with a loading rate of 100 mg/L applying a three-day period of magnetic stirring to ensure maximum dissolution of the test item in medium. The obtained mixture was allowed to settle for a period of one hour. Thereafter, the aqueous Saturated Solution (SS) was collected by means of siphoning and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
- Controls: Test medium without test item or other additives

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus 1820
- Source: In-house laboratory culture with a known history
- Age of parental stock: >2 weeks old
- Age at test initiation: <24 hours
- Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.

CULTIVATION
- Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of M7 medium in an all-glass culture vessel.
- Maximum age of the cultures: 4 weeks
- Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
- Temperature of medium: 18-22°C
- Feeding: Daily, a suspension of fresh water algae.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

180 mg/L (expressed as CaCO3)

Test temperature:

20 - 21°C

pH:

At t=0 h: 7.9 - 8.0
At t=48 h: 8.0 - 8.1

Dissolved oxygen:

At t=0 h: 8.2 - 8.4 mg/L
At t=48 h: 7.6 - 7.9 mg/L

Nominal and measured concentrations:

Nominal: 1.0, 1.8, 3.2, 5.6 and 10% of the SS prepared at a loading rate of 100 mg/L
Measured at the start of the test: were 0.83, 1.5, 2.6, 4.4 and 7.9 mg/L. The initial concentrations remained stable throughout the test, i.e. were at 97 – 100% relative to initial at the end of the test. Based on these results, effect parameters were expressed in terms of initially measured concentrations. See 'Any other information on results' for details on measured concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL, all-glass vessel, airtight closed without headspace
- Aeration: no aeration
- No. of organisms per vessel: 5 daphnids
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO medium, prepared with tap water purified by Reverse Osmosis.
- Culture medium different from test medium: Yes, M7 medium
- Intervals of water quality measurement: pH and dissolved oxygen: at the beginning and at the end of the test, for all concentrations and the control. Temperature of medium: continuously in a temperature control vessel, beginning at the start of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours photoperiod daily

EFFECT PARAMETERS MEASURED
- Immobility (including mortality): at 24 hours and at 48 hours

RANGE-FINDING STUDY
- Test concentrations: Control, 1.0, 10 and 100% of a SS prepared at a loading rate of 100 mg/L
- Results used to determine the conditions for the definitive study: Yes; the expected EC50 was between 1.0 and 10 mg/L (nominal loading rate).

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (performed Jul 2018)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Remarks on result:

other: 95% confidence interval: 2.0 - 2.4 mg/L

Effect concentrations 2

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

3.4 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Remarks on result:

other: 95% confidence interval: 2.6 - 4.4 mg/L

Details on results:

- Behavioural abnormalities: none observed in the Control group.
- Mortality of control: none
- Not more than 10% immobility was observed in the control (i.e. 5%), while no immobility was observed at the two lowest test concentrations throughout the test. At the end of the test, 85% of the daphnids exposed to 2.6 mg/L were recorded to be immobile. Complete immobility was observed at 4.4 and 7.9 mg/L already after 24 hours of exposure. See Table 2 in 'Any other information on results' for full details on immobility.
- Small responses at the retention time of the test item were detected in the chromatograms of the blank QC samples. The maximum contribution to the QC samples at the lowest concentration level was 6.4%. It was considered that it has no significant effect on the results of the test samples.
- All water quality parameters remained within the requirements as laid down in the study plan throughout the test.

Results with reference substance (positive control):

- Results with reference substance valid?
Yes
- The 48h-EC50 was 0.59 mg/L with a 95% confidence interval between 0.53 and 0.72 mg/L.
- The responses in this reference test with K2Cr2O7 are within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was between 0.3 and 1.0 mg/L. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at the Test Facility.

Reported statistics and error estimates:

The 48h-EC50 was calculated from the weibits of the percentages of affected daphnids and the logarithms of the corresponding test item concentrations (initially measured) using the maximum likelihood estimation method.
The 24h-EC50 could not be determined using a regression method. This was because there was no concentration between the highest concentration (A) at which 0% immobility and the lowest concentration (B) at which 100% immobility occurred. Instead, the EC50 was calculated as (AB)½, with A and B being limits of the 95% confidence interval.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Any other information on results incl. tables

Table 1: Final Test: Test Samples

Time of sampling [hours]	Percentage of SS^* [%]	Analyzed concentration [mg/L]	Relative to initial [%]
0	0	n.d.
	1.0	0.831
	1.8	1.50
	3.2	2.62
	5.6	4.37
	10	7.91
48	0	n.d.	n.a.
	1.0	0.808	97
	1.8	1.50	100
	3.2	2.56	98
	5.6	4.33	99
	10	7.83	99

*     Percentage of a saturated solution (SS) prepared at a loading rate of 100 mg/L.

n.d. Not detected.

n.a. Not applicable.

Table 2: Number of Introduced Daphnids and Incidence of Immobility in the Final Test 

Time (h)	Replicate	Initially measured conc. (mg/L)
		Control	0.83	1.5	2.6	4.4	7.9
0	A	5	5	5	5	5	5
	B	5	5	5	5	5	5
	C	5	5	5	5	5	5
	D	5	5	5	5	5	5
	Total introduced	20	20	20	20	20	20
24	A	0	0	0	0	5	5
	B	0	0	0	0	5	5
	C	0	0	0	0	5	5
	D	0	0	0	0	5	5
	Total immobilised	0	0	0	0	20	20
	Effect %	0	0	0	0	100	100
48	A	0	0	0	5	5	5
	B	1	0	0	3	5	5
	C	0	0	0	4	5	5
	D	0	0	0	5^#	5	5
	Total immobilised	1	0	0	17	20	20
	Effect %	5	0	0	85	100	100

^#Microscopic observation revealed that no test item was attached to the daphnids.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks' for details on validity criteria.

Conclusions:

The 48h-EC50 for Daphnia magna exposed to the test item was 2.2 mg/L based on initially measured concentrations (95% confidence interval between 2.0 and 2.4 mg/L).

Executive summary:

In a 48 h acute toxicity test conducted according to OECD guideline 202, Daphnia magna were exposed to solutions containing 1.0, 1.8, 3.2, 5.6 and 10% of a Saturated Solution prepared at a loading rate of 100 mg/L and an untreated control (all with 4 replicates per concentration and 5 daphnids per replicate). Measured concentrations were 0.83, 1.5, 2.6, 4.4 and 7.9 mg/L and remained stable throughout the test, i.e. were at 97 - 100% relative to initial at the end of the test. Therefore, effect parameters were based on initially measured concentrations. Not more than 10% immobility was observed in the control (i.e. 5%), while no immobility was observed at the two lowest test concentrations throughout the test. At the end of the test, 85% of the daphnids exposed to 2.6 mg/L were recorded to be immobile. Complete immobility was observed at the two highest test concentrations, already after 24 hours of exposure. The 48 -hEC50 for immobility was 2.2 mg/L (95% confidence interval 2.0 - 2.4 mg/L) based on initially measured concentrations. The study met all validity criteria and is considered to be reliable without restrictions.