Reaction mass of 6,6-dimethyl-2-methylidenebicyclo[3.1.1]heptane and rel-6,6-dimethylspiro[bicyclo[3.1.1]heptane-2,2'-oxirane]

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:

27 February 2017 - 9 April 2017

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:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Deviations:

no

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:

December 14, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control and 0.1, 0.32, 1.0, 3.2 and 10 mg/L
- Sampling method: samples (100 mL) were taken from the control and each test group from the freshly prepared bulk test preparation at 0 and 24 hours and from the pooled replicates at 24 and 48 hours.
- Sample storage conditions before analysis: test samples were analyzed on the day of receipt

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Nominal amounts of test item (2.2, 7.0 and 22 mg) were each separately added to the surface of 2.2 liters of test water in a test vessel which was sealed with minimal head space to give the 1.0, 3.2 and 10 mg/L loading rates respectively. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 1.0, 3.2 and 10 mg/L loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. Given the very low loading rates required, a series of dilutions was performed to give the remaining loading rates of 0.10 and 0.32 mg/L.
- Controls: test medium without test item
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no - At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Source: in-house laboratory culture
- Feeding during culture:
* Food type: mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension
* Frequency: daily
- Feeding during test: no
- Age at study initiation: young daphnids with an age of < 24 hours

ACCLIMATION
- Acclimation period: no

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room maintaining the water temperature at 18 to 22 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

none

Test temperature:

21 °C - 22 °C

pH:

fresh media: 7.5-7.7
used media: 7.5-7.6

Dissolved oxygen:

fresh media: 8.5-8.9 mg O2/L
used media: 8.3-8.9 mg O2/L

Nominal and measured concentrations:

RANGE-FINDING TEST
Two range-finding tests were performed at nominal loading rates of: 1.0, 10 and 100 mg/L (first test) and 0.1 and 1.0 mg/L (second test).
The measured test concentrations at 24 hours ranged from less than the limit of quantification, which was 0.0053 mg/L, to 18 mg/L. There was no significant change in the measured concentrations at 48 hours.

DEFINITIVE TEST
Based on the results of the range finding tests, the following nominal loading rates were assigned to the definitive test: 0.1, 0.32, 1.0, 3.2 and 10 mg/L.
The measured concentrations in fresh test preparations at 0 and 24 hours ranged from 0.042 to 2.0 mg/L, as total peak area. The measured concentrations inaged test preparations at 24 and 48 hours ranged from less than the limit of quantification, which was 0.0053 mg/L, to 0.60 mg/L, as total peak area.
Measured concentrations of Component 1 in the freshly prepared test concentrations at 0 and 24 hours were 0.017 - 1.1 mg/L, and in the old ones at 24 and 48 hours ranged from less than the limit of quantification, which was 0.0027 mg/L, to 0.14 mg/L.
Measured concentrations of Component 2 were less than the limit of quantification in all samples except for the 10 mg/L loading rate at 0 hours with a concentration of 0.011 mg/L.

Geometric mean measured test concentrations were used to calculate effect parameters in order to give a “worst case” analysis of the data.

For the total peak area, the geometric means were: 0.027, 0.11, 0.36, 0.54 and 1.0 mg/L.
For the Component 1, the geometric means were: 0.011, 0.056, 0.20, 0.30 and 0.50 mg/L.
The value of the Measured geometric mean concentration for Total Peak Area includes the Measured geometric mean concentration of Component 1 and other peaks that were detected (the test item is a multiple-component substance), but not the Component 2, because it was not detected in the test solutions.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL completely filled with test media and sealed to reduce losses through volatilisation
- Fill volume: approximately 100 mL (in principle no headspace as test vessels were completely filled)
- Aeration: no
- Renewal rate of test solution: yes, after 24 hours
- No. of organisms per vessel: 5
- 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 according to OECD 202
- Culture medium different from test medium: yes - culture medium: Elendt M7
- Intervals of water quality measurement: water temperature, pH and dissolved oxygen concentrations were recorded daily

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods
- Light intensity: 699 to 734 Lux
- Other: for renewal at 24 hours, the daphnids transferred by wide bore pipette from the 24-Hour old test media into the fresh test media

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mobility (at 24 and 48 hours)

RANGE-FINDING STUDY
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate (October 2016)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.56 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI not possible to determine

Details on results:

RANGE-FINDING TEST
- In the first range-finding test, all Daphnia in the 10 and 100 mg/L groups were found immobilized after 24 hours, while in the group of 1.0 mg/L none of the Daphnia was immobilized, but after 48 hours they were all pale.
- In the second range-finding test, in the group of 1.0 mg/L, after 24 hours all Daphnia were pale, and after 48 hours 6 were immobilized and the remaining 6 were pale and with reduced mobility.

DEFINITIVE TEST
- Observed sub-lethal effects: reduced movement was observed in the 3.2 mg/L solutions at 24 and 48 hours (13 Daphnia at both 24 and 48 hours); pale Daphnia were observed as well, in the group of 1.0 mg/L (10 Daphnia in total at 24 hours and 20 Dpahnia in total at 48 hours)
- Mortality of control: no
- Other adverse effects control: no
- Effect concentrations exceeding solubility of substance in test medium: no

Individual pH, temperature and dissolved oxygen values remained within acceptable limits throughout the duration of the study.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Test concentrations: 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L
- 48h-EC50: 1.2 mg/L

Reported statistics and error estimates:

The EL/EC50 values and associated confidence limits at 24 and 48 hours and the slope of the response curve and its standard error were calculated by Probit analysis using Linear Maximum-Likelihood regression. The Lowest Observed Effect Loading Rate (or Lowest Observed Effect Concentration) and the No Observed Effect Loading Rate (or No Observed Effect Concentration) at 24 and 48 hours were calculated using the Fisher’s Exact Binomial Test with Bonferroni correction.
All results were calculated using the ToxRat Professional computer software package (TOXRAT).

Table 1. Cumulative Immobilization Data and Observations in the Definitive Test

Nominal Concentration (% v/v Saturated Solution)		24 Hours
		Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)						Observations
		R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control		0	0	0	0	0	0	5 N	5 N	5 N	5 N
10		0	0	0	0	0	0	5 N	5 N	5 N	5 N
18		0	0	0	0	0	0	5 N	5 N	5 N	5 N
32		0	0	0	0	0	0	5 N	5 N	5 N	5 N
56		1	0	0	0	1	5	4 N	5 N	5 N	5 N
100		0	0	0	0	0	0	5 R	5 R	5 R	5 R

 

Nominal Concentration (% v/v Saturated Solution)		48 Hours
		Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)						Observations
		R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control		0	0	0	0	0	0	5 N	5 N	5 N	5 N
10		0	0	0	0	0	0	5 N	5 N	5 N	5 N
18		0	0	0	0	0	0	5 N	5 N	5 N	5 N
32		0	1	0	0	1	5	5 N	4 N	5 N	5 N
56		1	0	1	1	3	15	4 N	5 N	4 N	4 N
100		5	5	5	5	20	100	A/I	A/I	A/I	A/I

 

R1 – R4 = Replicates 1 to 4

N = No sub-lethal effects observed

R = Reduced movement

A/I = All daphnia immobilized

 

Verification of Test Concentrations

Chemical analysis of the freshly prepared test preparations at 0 and 24 hours (see Table 3 below) measured test concentrations to range from less than the LOQ to 0.76 mg/L. Analysis of the old or expired test preparations at 24 and 48 hours showed that measured test concentrations ranged from less than the LOQ to 0.73 mg/L, hence it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data.

The geometric mean measured test concentrations were determined to be:

 

Table 2. Geometric mean measured test concentrations

Nominal Test Concentration (% v/v Saturated Solution)	Geometric Mean Measured Test Concentration (mg/L)
10	0.10
18	0.10
32	0.24
56	0.38
100	0.70

Table 3. Test sample results

Time Point	Nominal Concentration of Test Item in Test Sample	Sample Preparation Factor	Determined Concentration of Test Item in Test Sample
	cnom	F	c
[hours]	[% v/v Saturated Solution]		[mg/L]
0 (Fresh)	Control	0.1	<LOQ
	10	0.1	0.0695
	18	0.1	0.173
	32	0.1	0.279
	56	0.1	0.465
	100	0.1	0.756
24 (Old)	Control	0.1	0.0681 (<LOQ)
	10	0.1	0.0905
	18	0.1	0.112
	32	0.1	0.227
	56	0.1	0.305
	100	0.1	0.659
24 (Fresh)	Control	0.1	<LOQ
	10	0.1	0.0748
	18	0.1	0.129
	32	0.1	0.242
	56	0.1	0.398
	100	0.1	0.665
48 (Old)	Control	0.1	0.0587 (<LOQ)
	10	0.1	<LOQ
	18	0.1	0.156
	32	0.1	0.229
	56	0.1	0.378
	100	0.1	0.728

LOQ = Limit of Quantification = 0.20 mg/L.

LOQ discussion: Recoveries were carried out at 0.1 mg/L, which corresponded to the concentration obtained for the 10% v/v saturated solution. The recoveries were repeated twice and gave an 89% recovery rate with an RSD of 15% (n = 5) and a 69% recovery rate with an RSD of 18% (n = 5). Due to the co-elution of peaks at the retention time of the test item, the recoveries were repeated at 0.2 mg/L and gave a recovery rate of 94% with an RSD of 3.0% (n = 5). The LOQ was therefore assessed as 0.20 mg/L. Sample concentrations at approximately 0.1 mg/L are recoverable, but with a greater degree of imprecision, and are reported for indicative purposes only.

Validity criteria fulfilled:

yes

Remarks:

1) In the control, no daphnids became immobilised or showed other signs of disease or stress. 2) The oxygen concentration at the end of the test was ≥3 mg/L in control and test vessels.

Conclusions:

The 48h-EC50 value to Daphnia magna was 0.56 mg/L based on geometric mean measured concentrations.

Executive summary:

A study was performed to assess the acute toxicity of the substance to Daphnia magna. The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 202 and GLP under semi-static conditions. Based on the results of a range-finding test, the following nominal concentrations were tested in the definitive test: untreated control, 0.1, 0.32, 1.0, 3.2 and 10 mg/L. For each concentration and a control group, twenty Daphnia magna (less than 24 hours old) were exposed for 48 hours in air-tight closed vessels in view of the high volatility of the substance It cannot be excluded that part of the substance volatilized into the headspace. The incidence of immobilisation was recorded for each test and control group at 24 hours and at 48 hours. Samples taken from all concentrations at the start and the end of both 24 -hour renewal periods were analysed with a validated GC-FID method. As the toxicity could not be related to one or the other constituent of Rosemarel, the total peak area was used to calculate the concentration of the substance. Since test concentrations did not remain stable during the 24 -hour renewal periods (i.e. not within 80 -120% of initial), geometric mean measured concentrations (0-48 hours) were calculated. The range tested based on geometric mean measured concentrations for the total peak area was: 0.027, 0.11, 0.36, 0.54 and 1.0 mg/L. The 48h-EC50 value to Daphnia magna was 0.56 mg/L based on geometric mean measured concentrations.

Description of key information

A study was performed to assess the acute toxicity of the substance to Daphnia magna. The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 202 and GLP under semi-static conditions. Based on the results of a range-finding test, the following nominal concentrations were tested in the definitive test: untreated control, 0.1, 0.32, 1.0, 3.2 and 10 mg/L. For each concentration and a control group, twenty Daphnia magna (less than 24 hours old) were exposed for 48 hours in air-tight closed vessels in view of the high volatility of the substance It cannot be excluded that part of the substance volatilized into the headspace. The incidence of immobilisation was recorded for each test and control group at 24 hours and at 48 hours. Samples taken from all concentrations at the start and the end of both 24 -hour renewal periods were analysed with a validated GC-FID method. As the toxicity could not be related to one or the other constituent of Rosemarel, the total peak area was used to calculate the concentration of the substance. Since test concentrations did not remain stable during the 24 -hour renewal periods (i.e. not within 80 -120% of initial), geometric mean measured concentrations (0-48 hours) were calculated. The range tested based on geometric mean measured concentrations for the total peak area was: 0.027, 0.11, 0.36, 0.54 and 1.0 mg/L. The 48h-EC50 value to Daphnia magna was 0.56 mg/L based on geometric mean measured concentrations.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.56 mg/L