Reaction mass of (2Z)-5-[(1R,3R,6S)-2,3-dimethyltricyclo[2.2.1.02,6]hept-3-yl]-2-methyl-2-penten-1-ol and (2Z)-2-methyl-5-[(1S,2R,4R)-2-methyl-3-methylenebicyclo[2.2.1]hept-2-yl]-2-penten-1-ol

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:

From 14 January 2020 to 28 January 2020

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:

Inspected on 29-30-31 May 2018 / Signature date: 15 November 2018

Analytical monitoring:

yes

Details on sampling:

Single samples for analysis were taken from the control and all test solutions at the start (t=0 h), at t=24 h (new and old solutions) and at the end of the test (t=48 h).
Chemical analyses were performed on the two major constituents of the test item and used as tracers to provide an indication of the concentration of dissolved organic material in the WAFs and stability.

Vehicle:

no

Details on test solutions:

- Preparation of stock Water Accommodated Fractions (WAFs): The study was carried out using WAFs. The WAFs (for fresh media at t=0h and t=24h) were prepared under closed conditions and by slow-stirring to avoid production of a dispersion. The mixing vessels were cylindrical glass bottles sealed with screw caps and fitted with a drain port near the bottom for drawing off the WAFs. The volume of each mixing vessel was approximately 5 L. A magnetic stirring bar was placed in each test vessel and 5.2 L to 5.7 L of test water (depending on the brim capacity of the bottles) were added in order to use a maximum volume and to minimise headspace. The loading rates of the test item were weighed on glass slides that afterwards were placed under the surface of the test water contained in the mixing vessels through fishing wire. Then the mixing vessels were closed immediately. The mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. After 24 ± 2 hours of gentle stirring in the dark at room temperature, the WAFs were allowed to stand undisturbed for at least 1 hour before use. The first 100 mL were discarded via the drain port. Then the WAFs were directly added into test vessels that were immediately sealed with screw caps after introduction of daphnids. No small bubble was observed in the test vessels. The test solutions in test vessels were observed to be clear and colourless at all loading rates. The Tyndall effect (checked via laser beam) was negative in all treatments (for fresh media at t=0h and t=24h). The test was carried out without adjustment of the pH.
- Test concentrations: Based on the results of range-finding tests, test solutions used in the definitive test were prepared to obtain the following nominal loading rates (spaced by a factor of 1.50): 0.30, 0.44, 0.67, 1.0 and 1.5 mg/L
- Controls: Test water without test substance but treated in the same way as the test substance solutions.

Test organisms (species):

Daphnia magna

Details on test organisms:

- Species: Daphnia magna (Straus), clone 5
- Sex: Female
- Origin: LIEBE - CNRS UMR 7146 - UFR SciFA - Université de Lorraine Campus Bridoux - Bât. IBISE, 8, rue du Général Delestraint - 57070 METZ, bred in the Laboratoires des Pyrénées et des Landes.
- Reason for selection: Characteristic and common representative of freshwater zooplankton which has been selected as an internationally accepted invertebrate species.
- Validity of batch: Daphnids originated from a healthy stock, showing no signs of stress such as mortality, presence of males, ephippia or discoloured Daphnids.
- Age at test start: < 24 hours old
- Breeding conditions: Daphnids were cultured in the Laboratoires des Pyrénées et des Landes under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in all-glass vessel containing test water. Cultures were maintained at a density of 1 adult daphnid per 25 mL of culture medium. Daphnids were fed at least three times a week with a suspension of algal cells (Pseudokirchneriella subcapitata) up to 0.1-0.2 mg C.Daphnia.-1day.-1. The water was changed three times a week. These culture conditions maintained the daphnids in the parthenogenetic reproductive stage.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

none

Hardness:

No data

Test temperature:

The temperature of the test medium was situated between 20.6 and 21.1°C throughout the test (average value: 20.9°C), and complied with the requirements as laid down in the study plan (20°C ± 2°C, constant within 1°C)

pH:

pH: 6.0-9.0, not varying by more than 1.5 units.
See table 6.1.3/1 in "Any other information on results incl. tables".

Dissolved oxygen:

Oxygen: ≥ 60% of the air-saturation value at the end of the test.
See table 6.1.3/2 in "Any other information on results incl. tables".

Salinity:

Not applicable

Conductivity:

No data

Nominal and measured concentrations:

- Nominal Loading Rates: 0.30, 0.44, 0.67, 1.0 and 1.5 mg/L
- Measured concentrations: See tables 6.1.3/4 and 5 in "Any other information on results incl. tables".

Details on test conditions:

TEST SYSTEM
- Test type: Semi-static (24h renewals), in order to prevent the dissipation of the test item from test solutions because of its volatility and the expected degradation of some constituents, and to maintain dissolved O2 concentrations as far as possible; at each renewal, a new series of clean and sterile test vessels was used.
- Test vessels: 60-mL glass flasks sealed with assembled screw cap with hole and PTFE/silicone septum. Each test vessel was uniquely identified with study code, replicate number, date of experimentation and treatment group.
- Number of daphnids: 20 per treatment group (including controls), divided into 4 groups of 5 daphnids
- Loading: 5 daphnids per vessel each completely filled with test solution and without headspace
- Number of replicates: 4 replicates with daphnids per treatment group.
- Aeration: No aeration of the test solutions occurred throughout the test.
- Feeding: no feeding.
- Introduction of daphnids: Daphnids were introduced into the test vessels each completely filled with test solution and stoppered without headspace immediately after filling the test vessels with test solutions.

TEST WATER
Reconstituted water (Elendt M4 medium), as prescribed in OECD Guideline 202. The pH of this solution was in the range of 6 to 9 and the total water hardness was approximately 250 mg.L-1 (as CaCO3).

OTHER TEST CONDITIONS
- Test environment: Controlled environment cabinet (20°C ± 2°C)
- Light regime: 16h light : 8h dark

EFFECT PARAMETERS MEASURED:
- Immobility: Immobility and abnormal behaviour were determined by visual observation after 24 and 48 hours. Daphnids were considered to be immobile if they were not able to swim within 15 seconds after gentle agitation of test vessels. The evaluation of the effects was based on the nominal WAFs concentrations (nominal loading values). The software ToxRat® Professional was used for the determination of the effective concentrations.
- pH and dissolved O2: At start (t=0 h), at t=24 h (new and old solutions) and at the end of the test (t=48 h) from all treatment groups.
- Temperature of medium: At start (t=0 h), at t=24 h (new and old solutions) and at the end of the test (t=48 h) from all treatment groups.

RANGE-FINDING STUDY
- Loading Rates: 0.32, 1.0, 3.2, 10.0, 32.0 and 100.0 mg/L
- Results used to determine the conditions for the definitive study: 0% immobilisation was observed in the control and at 0.32 mg/L after 24 and 48 hours of exposure. However, 100% immobilisation was observed at 1.0, 3.2, 10.0, 32.0 and 100.0 mg/L after 24 and 48 hours of exposure. The results of the turbidity analysis performed at t=0h suggested that no dissolved material was present in the WAFs. Further to this range-finding test, a complementary test was performed in order to be sure about the effects observed and finalise the study plan. Different conditions were tested: extension of settling period (1h ‘classique”; 4h or 24h) after the slow stirring phase; filtered or not the test solutions (with a 0.2 µm cellulose acetate membrane filter); look at the homogeneity of the solutions in terms of concentration (by measured analytically aliquot at the bottom, in the middle and at the top of the vial); observation of solutions and turbidity analysis. These results suggest that the longer the settling period of WAFs, the less effect there is on daphnia; but this was certainly due to the resulting loss of the test item. In addition, measured concentrations of each constituent at 1 mg/L - 1h settling period - (after sampling in the test vessels in 3 different areas) were very close to their expected nominal concentration, and immobilisation results were consistent with those from the range-finding test. Therefore, it was decided to carry out the definitive test using WAFs stirred for 24h, and rested for about 1 hour.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

0.612 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 0.548-0.682 mg/L

Details on results:

- Analytical results: See tables 6.1.3/4 and 5 in "Any other information on results incl. tables". The analytical results of this test showed that WAFs were overall stable throughout the test period. Indeed, analytical monitoring of the two major constituents of the test item revealed that their concentrations were satisfactorily maintained within or very close to ± 20% of the initial concentration between the start and the end of each exposure period (t=0h-t24hOld and t=24hFresh-t=48h). Besides, these concentrations were satisfactorily maintained within ± 20% of their expected nominal concentrations throughout the test. Therefore, and since the test item was a multi-constituent substance, the results were based on nominal loading rates and as Effective Loading Rate 50, according to the OECD No. 23
- Biological results: See table 6.1.3/3 in "Any other information on results incl. tables". After 48 hours of exposure, immobilisation rates were 0% at 0.30 mg/L, 10% at 0.44 mg/L, 60% at 0.67 mg/L and 100% at 1.00 mg/L and 1.50 mg/L. Therefore, the highest loading rate without observed effect after 48 h was 0.30 mg/L.

Results with reference substance (positive control):

On November 7, 2019 (KD19-003; most recent test), the 24h-EC50 was 1.243 mg/L. Hence, the sensitivity of the clone of Daphnia magna was in agreement with OECD 202 (expected 24h-EC50: 0.6 mg/L to 2.1 mg/L) at this time.

Reported statistics and error estimates:

The EL50 values including the 95% confidence interval using probit analysis were determined by the computer program ToxRat

Table 6.1.3/1: pH-values during the final test

		Nominal concentration*(mg test item/L)
		Control	0.30	0.44	0.67	1.00	1.50
Start t=0 h		8.81	8.69	8.64	8.65	8.66	8.67
t=24 h	Old	8.52	8.49	8.56	8.58	8.59	8.63
	Fresh	8.47	8.58	8.56	8.59	8.61
End t=48 h		8.54	8.63	8.57	8.63	8.62

* WAF prepared at the given loading rate.

Grey boxes: no further analysis was performed for concentrations in which all daphnids were immobilised.

Table 6.1.3/2: Dissolved oxygen concentration (mg/L) during the final test

		Nominal concentration*(mg test item/L)
		Control	0.30	0.44	0.67	1.00	1.50
Start t=0 h		8.65	8.59	8.68	8.67	8.60	8.60
t=24 h	Old	8.46	8.47	8.51	8.52	8.38	8.25
	Fresh	8.58	8.42	8.50	8.35	8.60
End t=48 h		8.31	8.48	8.50	8.42	8.32

* WAF prepared at the given loading rate.

Grey boxes: no further analysis was performed for concentrations in which all daphnids were immobilised.

All these dissolved-oxygen concentrations correspond to values > 90% of the air-saturation value.

Table 6.1.3/3: Acute immobilisation of daphnids after 24 and 48 hours in the final test

Nominal concentration*(mg test item/L)	Replicate	Number of daphnids exposed	Immobilisation at 24 h		Immobilisation at 48 h
			Number	Total %	Number	Total %
Control	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
0.30	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
0.44	1 2 3 4	5 5 5 5	0 0 0 0	0	1 0 0 1	10
0.67	1 2 3 4	5 5 5 5	0 2 2 2	30	3 4 3 4	60
1.00	1 2 3 4	5 5 5 5	5 2 3 3	75	5 5 5 5	100
1.50	1 2 3 4	5 5 5 5	5 5 5 5	100	5 5 5 5	100

* WAF prepared at the given loading rate.

Table 6.1.3/4: Concentration of the test item (mg/L) in test water in the final test - Constituent 1

Nominal concentration* (mg/L)	Expected nominal concentration in consituent 1 (mg constituent 1/L)	Measured concentration in consituent 1 (mg constituent 1/L)						Geometric mean measured concentrations
		Start (t=0h)	t=24hold	Relative loss to initial value (t=0h - t=24hold) (%)	t=24hfresh	End t=48h	Relative loss to initial value (t=24hfresh- t=48h) (%)	mg/L	% nominal
Control	0	Abs.	Abs.	N.A.	Abs.	Abs.	N.A.	N.A.	N.A.
0.30	0.16	0.16	0.17	-6	0.18	0.16	11	0.17	106
0.44	0.24	0.21	0.22	-5	0.26	0.24	8	0.23	96
0.67	0.36	0.32	0.28	13	0.38	0.33	13	0.33	92
1.00	0.54	0.53	0.53	0	0.54	0.46	15	0.51	94
1.50	0.82	0.79	0.77	3				0.78	95

* WAF prepared at the given loading rate

N.A.: not applicable

% = Percent of expected nominal concentration in Constituent 1.

Grey boxes: no further analysis was performed for concentrations in which all daphnids were immobilised.

Abs.= Absence: concentrations below the LOQ (0.11 mg/L) and the LOD (0.03 mg/L).

Table 6.1.3/5: Concentrations of the test item (mg/L) in test water in the final test - Constituent 2

Nominal concentration* (mg/L)	Expected nominal concentration in consituent 2 (mg constituent 2/L)	Measuredconcentration in constituent 2 (mg constituent 2/L)						Geometric mean measured concentrations
		Start (t=0h)	t=24hold	Relative loss to initial value (t=0h - t=24hold) (%)	t=24hfresh	End t=48h	Relative loss to initial value (t=24hfresh- t=48h) (%)	mg/L	% nominal
Control	0	Abs.	Abs.	N.A.	Abs.	Abs.	N.A.	N.A.	N.A.
0.30	0.08	0.07	0.08	-14	0.09	0.07	22	0.08	100
0.44	0.11	0.09	0.10	-11	0.12	0.11	8	0.10	91
0.67	0.17	0.14	0.13	7	0.18	0.15	17	0.15	88
1.00	0.26	0.24	0.24	0	0.25	0.21	16	0.23	88
1.50	0.38	0.37	0.36	3				0.36	95

* WAF prepared at the given loading rate

N.A.: not applicable

% = Percent of expected nominal concentration in Constituent 2.

Grey boxes: no further analysis was performed for concentrations in which all daphnids were immobilised.

Abs.= Absence: concentrations below the LOQ (0.05 mg/L) and the LOD (0.02 mg/L).

Validity criteria fulfilled:

yes

Remarks:

In the control, no daphnids became immobilised nor trapped at the surface of the water nor showed signs of stress. In addition, dissolved oxygen concentration at the end of the test was ≥ 60% of the air-saturation value in controls and test vessels.

Conclusions:

The toxic effect of the test substance to the freshwater invertebrate Daphnia magna was investigated in a closed semi-static test using Water Accommodated Fractions. Under experimental conditions, and based upon nominal loading rates, the 48-hour EL50 value was estimated to be 0.612 mg/L (95% CL: 0.548 -0.682 mg/L).

Executive summary:

This GLP study was performed to assess the acute toxicity of test item to Daphnia magna. The method followed was designed to be compliant with OECD Guideline 202, referenced as EU Method C.2 and with the “Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals” (OECD No. 23). The criterion measured was the 48 h EL50 (Median Effective Loading rate), a statistically derived loading rate which is expected to cause immobility in 50% of the daphnids within a period of 48 hours.

Based on the results of range-finding tests, twenty daphnids (four replicates, five daphnids per replicate) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading values of 0.30, 0.44, 0.67, 1.0 and 1.5 mg/L, and to a control. The immobilisation of the daphnids was determined in a closed semi-static 48-hour test by visual observation after 24 and 48 hours. The concentrations of the test item, represented by analytical follow-up of the two main constituents, were determined at the start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h).

Analytical monitoring of the two major constituents of the test item revealed that their concentrations were satisfactorily maintained within or very close to ± 20% of the initial concentration between the start and the end of each exposure period (t=0h-t24hOld and t=24hFresh-t=48h), suggesting that WAFs were overall stable throughout the test. After 48 hours of exposure, immobilisation rates were 0% at 0.30 mg/L, 10% at 0.44 mg/L, 60% at 0.67 mg/L and 100% at 1.00 mg/L and 1.50 mg/L.

Under experimental conditions, and based upon nominal loading rates, the 48-hour EL50 value was estimated to be 0.612 mg/L (95% CL: 0.548 -0.682 mg/L)

Description of key information

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

48h-EL50 (Daphnia magna) = 0.612 mg/L (95% CL: 0.548 - 0.682 mg/L)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.612 mg/L

Additional information

One key study is available to assess the short-term toxicity of the registered substance to aquatic invertebrates, Daphnia magna.

This study (LPL, 2020) was performed according to OECD Guideline 202 and EU Method C.2 with GLP compliance. Based on the results of range-finding tests, twenty daphnids (four replicates, five daphnids per replicate) were exposed to Water Accommodated Fractions (WAFs) of the registered substance over a range of nominal loading values of 0.30, 0.44, 0.67, 1.0 and 1.5 mg/L, and to a control. The immobilisation of the daphnids was determined in a closed semi-static 48 -hour test by visual observation after 24 and 48 hours. The concentrations of the test item, represented by analytical follow-up of the two main constituents, were determined at the start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h). Analytical monitoring of the two major constituents of the test substance revealed that their concentrations were satisfactorily maintained within or very close to ± 20% of the initial concentration between the start and the end of each exposure period, suggesting that WAFs were overall stable throughout the test. After 48 hours of exposure, immobilisation rates were 0% at 0.30 mg/L, 10% at 0.44 mg/L, 60% at 0.67 mg/L and 100% at 1.00 mg/L and 1.50 mg/L. Therefore, under experimental conditions, and based upon nominal loading rates, the 48-hour EL50 value was determined to be 0.612 mg/L (95% CL: 0.548 - 0.682 mg/L)