3a,4,5,6,7,7a-hexahydro-5-methoxy-4,7-methano-1H-indene

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:

14 October 2018 to 12 December 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)

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals, OECD series on testing and assessment number 23

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below:
- Frequency: at t = 0 h and t = 48 h
- Volume: 6.0 mL from the approximate centre of the test vessels
- Storage: Samples were stored in a 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, instead samples were taken from one test vessel.
Additionally, reserve samples of 6.0 mL were taken for possible analysis. If not used, these samples were stored in a freezer (≤-15 °C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The batch of test material tested was a clear colourless to light yellow liquid and not completely soluble in test medium at the loading rate initially prepared. No correction was made for the purity/composition of the test material. All glassware used during the preparation of test solutions was closed with minimal headspace in order to minimise vaporisation of the test material.
Preparation of test solutions started with a loading rate of 100 mg/L applying a one-day period of magnetic stirring to ensure maximum dissolution of the test material 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 colourless at the end of the preparation procedure. Any residual volumes were discarded.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain/clone: Straus, 1820
- Source: In-house laboratory culture with a known history; at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions. 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.
- Age: Daphnia, less than 24 hours old, from parental daphnids of more than two weeks old.
- Feeding during test: no

BREEDING
- Start of each batch: Approximately 250 newborn daphnids, i.e. less than 3 days old, were placed into 5 litres of 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.
- Culture medium: M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33). The following salts and vitamins were added to freshly prepared test medium to reach the following concentrations:
Salts: H3BO3 0.71 mg/L, FeSO4.7H2O 0.25 mg/L, MnCl2.4H2O 0.090 mg/L, LiCl 0.076 mg/L, RbCl 0.018 mg/L, SrCl2.6H2O 0.038 mg/L, Na2MoO4.2H2O 0.015 mg/L, NaBr 0.0040 mg/L, CuCl2.2H2O 0.0042 mg/L, ZnCl2 0.013 mg/L, CoCl2.6H2O 0.010 mg/L, KI 0.0032 mg/L, Na2SeO3 0.0022 mg/L, NH4VO3 0.00057 mg/L, Na2EDTA.2H2O 0.62 mg/L, Na2SiO3.5H2O 7.5 mg/L, NaNO3 0.27 mg/L, KH2PO4 0.14 mg/L and K2HPO4 0.18 mg/L.
Vitamins: Thiamine hydrochloride 75.0 µg/L, B12 1.0 µg/L and Biotin 0.75 µg/L.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

180 mg/L as CaCO3

Test temperature:

19 - 20 °C

pH:

7.9 - 8.1

Dissolved oxygen:

8.6 - 9.3 mg/L

Nominal and measured concentrations:

- Nominal: Control, 10, 18, 32, 56 and 100 % of the SS, prepared at a loading rate of 100 mg/L
- Measured: Control, 8.0, 15, 25, 46 and 84 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL, all-glass, airtight closed without headspace
- Material, size, headspace, fill volume: 60 mL
- Aeration: No aeration of the test solutions was applied
- No. of organisms per vessel: 5; Daphnids were introduced 17 minutes after preparation of the test solutions
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The following salts (analytical grade) were added to tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands): CaCl2.2H2O 211.5 mg/L, MgSO4.7H2O 88.8 mg/L, NaHCO3 46.7 mg/L and KCl 4.2 mg/L. The hardness of test medium expressed as CaCO3 was 180 mg/L with a pH between 6 and 9.
- Intervals of water quality measurement: pH and dissolved oxygen were measured at the beginning and at the end of the test, for all concentrations and the control. Temperature of medium was measured continuously in a temperature control vessel, beginning at the start of the test.

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

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

RANGE-FINDING STUDY
- The project started with a combined limit/range-finding test. Twenty daphnids per concentration (four replicates, 5 daphnids per vessel) were exposed to a control and a SS prepared at a loading rate of 100 mg/L. Test procedure and conditions were similar to those applied in the final test with the following exceptions: Ten daphnids per concentration (in duplicate, 5 per vessel) were exposed to 1.0 and 10 % of the SS prepared at a loading rate of 100 mg/L in the combined range-finding test and dissolved oxygen concentrations and pH were only measured in the control and the highest test concentration.
- Results used to determine the conditions for the definitive study: Yes

ACCEPTABILITY OF THE TEST
1. In the control, no daphnids became immobilised or showed other signs of disease or stress, for example discoloration or unusual behaviour such as trapping at the surface of the medium.
2. The dissolved oxygen concentration at the end of the test was ≥3 mg/L in control and test vessels.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

19 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 %-confidence interval: 18 - 21 mg/L

Details on results:

COMBINED LIMIT/RANGE-FINDING TEST
At the end of the test, no immobility was observed at the two lowest test concentrations and in the control. At the highest test concentration, 100 % immobility was observed after 24 hours of exposure. Therefore, the expected EC50 was between 10 and 100 % of the SS.
Based on these results, samples taken from solutions containing 10 and 100 % of the SS prepared at a loading rate of 100 mg/L were analysed. The measured concentrations at the start of the test were 9.2 and 94 mg/L, respectively. During the exposure period, the concentrations remained stable, i.e. were at 91 - 92 % relative to initial at the end of the test.
All test conditions were maintained within the limits prescribed by the study plan.

FINAL TEST
- Measured Concentrations: Samples taken from all test concentrations and the control were analysed. The measured concentrations at the start of the test were 8.0, 15, 25, 46 and 84 mg/L, respectively. During the exposure period, the concentrations remained stable, i.e. were at 96 - 102 % relative to initial at the end of the test. It should be noted that a response was measured in the control. However, it was most likely a result of carry over and therefore has no impact on the study results. The contribution of this response to the lowest concentration was 0.033 and 0.034 % at the start and end of the exposure period, respectively. Based on these results, effect parameters were expressed as initially measured concentrations.
- Immobility: No immobility was observed in the control and the two lowest test concentrations during the exposure period. At 24 hours of exposure a dose-response was observed at concentrations of 25 - 84 mg/L. At the end of the test all daphnids were immobilised at the three highest exposure concentrations. The responses recorded in this test allowed for reliable determination of an EC50 and were in agreement with what was expected based on the results of the combined limit/range-finding test.

ACCEPTABILITY OF THE TEST
- In the control, no daphnids became immobilised or showed other signs of disease or stress, for example discoloration or unusual behaviour such as trapping at the surface of the medium.
- The dissolved oxygen concentration at the end of the test was ≥3 mg/L in control and test vessels.

Results with reference substance (positive control):

- Daphnia were exposed for a maximum of 48 hours to K2Cr2O7 concentrations of 0.10, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/L and to a control. Twenty daphnids were exposed per concentration.
- The study met the acceptability criteria prescribed by the study plan and was considered valid.
- The actual responses in this reference test with K2Cr2O7 are generally within the ranges of the expected responses at the different concentrations, i.e. the 48 h-EC50 was between 0.28 and 0.90 mg/L. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at the testing facility.
- The 48 h-EC50 was 0.40 mg/L with a 95 % confidence interval between 0.33 and 0.48 mg/L.

Reported statistics and error estimates:

The 24 h and 48 h-EC50-values were calculated from the probits of the percentages of affected daphnids and the logarithms of the corresponding test material concentrations (initially measured) using the maximum likelihood estimation method. ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Table 1: Number of Introduced Daphnids and Incidence of Immobility in the Final Test 

Time (h)	Replicate	Test Material Measured conc. (mg/L)
		Control	8.0	15	25	46	84
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	4	4	5
	B	0	0	0	3	4	5
	C	0	0	0	3	4	5
	D	0	0	0	3	5	5
	Total immobilised	0	0	0	13	17	20
	Effect %	0	0	0	65	85	100
48	A	0	0	0	5^#	5	5
	B	0	0	0	5	5	5
	C	0	0	0	5	5	5
	D	0	0	0	5	5	5
	Total immobilised	0	0	0	20	20	20
	Effect %	0	0	0	100	100	100

^#Microscopic observation revealed no test material attached to the daphnids.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the 48 h-EC50 for Daphnia magna exposed to the test material was 19 mg/L based on measured concentrations (95 % confidence interval between 18 and 21 mg/L).

Executive summary:

The short-term toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guideline OECD 202 under GLP conditions.

The objective of the study was to evaluate the test material for its ability to generate acute toxic effects on the mobility of Daphnia magna during an exposure period of 48 hours and, if possible, to determine the EC50 at 24 and 48 hours of exposure.

A Saturated Solution (SS) of the test material was prepared at a loading rate of 100 mg/L and used as the highest concentration. Lower concentrations were prepared by diluting the highest concentration in test medium. All glassware used during the preparation of test solutions was closed with minimal headspace in order to minimise vaporisation of the test material.

A final test was performed based on the results of a combined limit/range-finding test. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to solutions containing 10, 18, 32, 56 and 100 % of the SS prepared at a loading rate of 100 mg/L. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

Samples taken from all test concentrations and the control were analysed. The measured concentrations at the start of the test were 8.0, 15, 25, 46 and 84 mg/L, respectively. During the exposure period, the concentrations remained stable, i.e. were at 96 - 102 % relative to initial at the end of the test. Therefore, effect parameters were expressed in terms of measured concentrations.

No immobility was observed in the control and the two lowest test concentrations during the exposure period. At 24 hours of exposure a dose-response was observed at concentrations of 25 – 84 mg/L. At the end of the test all daphnids were immobilised at the three highest exposure concentrations.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

Under the conditions of this study, the 48 h-EC50 for Daphnia magna exposed to the test material was 19 mg/L based on measured concentrations (95 % confidence interval between 18 and 21 mg/L).

Description of key information

Under the conditions of this study, the 48 h-EC50 for Daphnia magna exposed to the test material was 19 mg/L based on measured concentrations (95 % confidence interval between 18 and 21 mg/L).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

19 mg/L

Additional information

The short-term toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guideline OECD 202 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The objective of the study was to evaluate the test material for its ability to generate acute toxic effects on the mobility of Daphnia magna during an exposure period of 48 hours and, if possible, to determine the EC50 at 24 and 48 hours of exposure.

A Saturated Solution (SS) of the test material was prepared at a loading rate of 100 mg/L and used as the highest concentration. Lower concentrations were prepared by diluting the highest concentration in test medium. All glassware used during the preparation of test solutions was closed with minimal headspace in order to minimise vaporisation of the test material.

A final test was performed based on the results of a combined limit/range-finding test. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to solutions containing 10, 18, 32, 56 and 100 % of the SS prepared at a loading rate of 100 mg/L. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

Samples taken from all test concentrations and the control were analysed. The measured concentrations at the start of the test were 8.0, 15, 25, 46 and 84 mg/L, respectively. During the exposure period, the concentrations remained stable, i.e. were at 96 - 102 % relative to initial at the end of the test. Therefore, effect parameters were expressed in terms of measured concentrations.

No immobility was observed in the control and the two lowest test concentrations during the exposure period. At 24 hours of exposure a dose-response was observed at concentrations of 25 – 84 mg/L. At the end of the test all daphnids were immobilised at the three highest exposure concentrations.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

Under the conditions of this study, the 48 h-EC50 for Daphnia magna exposed to the test material was 19 mg/L based on measured concentrations (95 % confidence interval between 18 and 21 mg/L).