Reaction mass of (3E)-4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one and 4-(dodecylsulfanyl)-4-(2,6,6-trimethylcyclohex-1-en-1-yl)butan-2-one and 4-(dodecylsulfanyl)-4-(2,6,6-trimethylcyclohex-2-en-1-yl)butan-2-one

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:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. Minor deviations not considered to impact the reliability of the study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

no positive reference control; seperation factor slightly above guideline preference (2.2)

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

yes

Remarks:

see above

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Deviations:

yes

Remarks:

see above

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No 8147, Agricultural Production Bureau, November 24 2000.

Deviations:

yes

Remarks:

see above

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 (2000)

Deviations:

not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: February 2014; signature: April 2014

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All loading levels and the control were analytically verified via GC-MS at the start (0 and 24 hours) and at the end of the exposure intervals (24 and 48 hours). Test substance loadings were prepared as Water Accommodated Fraction (WAF).
- Sampling method: The analysis described within this report was the analysis of one of the constituents measured at a specific wavelength.
The test concentrations of tracer constituent were measured using gas chromatography with mass spectrometric detection system (GC-MS). At the start of the definitive test, two samples (50 mL) were taken from the freshly-prepared control and test media. After 48 hours, the contents of the replicate flasks for each group were pooled and further samples taken for analysis. This was to assess the stability of the WAF.
- Sample storage conditions before analysis: All original samples were stored at room temperature before preparation and a separate sample stored frozen if required.

Vehicle:

no

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphinds
- Strain: Daphnia magna (Straus)
- Source: National Institute for Applied Chemical Research (IRCHA), France
- Age at study initiation (mean and range, SD): < 24 hours.
- Method of breeding: Parthenogenesis
- Feeding during test: No.
- Food type: Cultures were fed daily with a suspension of the unicellular green algae, Pseudokirchneriella subcapitata, to provide nominally 0.1 to 0.2 mg carbon per daphnid, per day, except during the initial two days when a slightly lower ration was given.
- Amount: See above.
- Frequency: See above. No feeding during test conditions.

ACCLIMATION
- Acclimation period: None.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

The adjusted-ISO medium M7 hardness: 168 mg/L expressed as CaCO3 and the pH: 7.8 ± 0.1

Test temperature:

The temperature continuously measured in a temperature control vessel varied between 20.0 and 21.0°C during the test, and complied with the requirements as laid down in the guideline (18-22°C, constant within 2°C).

pH:

Test conditions remained within the limits prescribed by the guideline; pH: 7.6-8.2 not varying by 1.5 units the end of the test. Actual pH: 7.8 ± 0.1

Dissolved oxygen:

Test conditions remained within the limits prescribed by the guideline; oxygen:>3 mg/L at the end of the test. Actual oxygen: start: 8.6 ± 0.25 mg/L and end: 9.2 ± 0.2 mg/L

Nominal and measured concentrations:

Range finding tests were performed prior to the definitive test: Information is provided in table 1. at concentrations 0 (control), 1, 10 and 100 mg/L.
Definitive test: nominal concentrations:
Five water accommodated fractions (WAF) were prepared with nominal loadings of the test item in the range: 0 (control), nominal loading rates of 1.94, 4.27, 9.39, 20.7, 45.5 and 100 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 120 ml
- Type (delete if not applicable): loosely covered; static.
- Material, size, headspace, fill volume: Glass, 100 ml medium (therefore headspace ca. 20 ml) in the definitive study
- Aeration: No aeration of the test solutions.
- No. of organisms per vessel: Control and test item: 20 per concentration, 5 per vessel containing 100 ml of test solution (definitive test)
- No. of vessels per concentration (replicates): 4 (four replicates, 5 daphnia per vessel).
- No. of vessels per control (replicates): 4 (four replicates, 5 daphnia per vessel).
- No. of vessels per vehicle control (replicates): Not applicable.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised water; used to prepare softened-ISO Medium M4. The following macro salts and micro elements were diluted in water and added to ISO Medium M4:
Microelements (mg/L): H3BO3 2.86 ; MnCl2.4H2O 0.36 ; LiCl 0.31 ; RbCl 0.071 ; SrCl2.6H2O 0.152 ; NaBr 0.016 ; Na2MoO4.2H2O 0.063 ; CuCl2 2H2O 0.017 ; ZnCl2 0.013 ; CoCl2.6H2O 0.010 ; KI 0.0033 ; Na2SeO3 0.0022 ; NH4VO3 0.00058 ; Fe-EDTA solution 3.50; Macro salts (mg/L): CaCl2.2H2O 147 ; MgSO4.7H2O 61.5 ; KCl 5.80 ; NaHCO3 64.8 ; Na2SiO3.9H2O 6.83 ; NaNO3 0.274 ; KH2PO4 0.143 ; K2HPO4 0.184 and Vitamins (mg/L): Thiamine hydrochloride 0.075 ; Cyanocobalamine (Vitamin B12) 0.0010 ; Biotin (Vitamin H) 0.00075 respectively
- Culture medium different from test medium: No, softened-ISO Medium M4 (above), renewed by three times per week.

OTHER TEST CONDITIONS
- Adjustment of pH: None.
- Photoperiod:16 hours photoperiod daily; 8 hours dark. With periods of subdued lighting at the beginning and end of each light phase.
- Light intensity: Not reported.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobility (including mortality), 24 hours and at 48 hours.

Reference substance (positive control):

no

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

25.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 19.60 -32.90 mg/L; nominal based on WAF

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

10.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 7.98 - 14.80 mg/L; nominal based on WAF

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

1.94 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: None
- Observations on body length and weight: Not applicable.
- Other biological observations:
- Mortality of control: No mortalities.
- Other adverse effects control: None.
- Abnormal responses: None.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None.
- Effect concentrations exceeding solubility of substance in test medium: No.

Reported statistics and error estimates:

Statistical analysis was performed using the SAFEStat LD50 application (version 1.5), SAS 9.1.3 (SAS Institute, 2002). Test results were expressed in terms of the nominal loading rates (EL50).
The “no observed effect loading rate” (NOELR) was derived by direct inspection of the data and based on the 10% effect seen at the end of the test.

Table 1. Environmental concentrations during the final test

Nominal loading rate of the test substance	Measured tracer constituent #	Temperature		pH		Dissolved Oxygen (%ASV)		Dissolved Oxygen (mg/L)
(mg/L)		0 h	48 h	0 h	48 h	0 h	48 h	0 h	48 h
Control	nd	21.2	20.7	7.70	7.72	98	102	8.80	9.25
1.94	1.26	21.2	20.7	7.81	7.73	97	100	8.67	9.04
4.27	2.40	21.2	20.8	7.85	7.75	96	100	8.54	9.05
9.39	5.71	21.1	20.8	7.87	7.77	98	100	8.79	9.02
20.7	10.2	21.1	20.8	7.86	7.75	99	104	8.84	9.38
45.5	23.6	21.0	20.8	7.89	7.77	97	104	8.73	9.40
100	46.3	21.0	20.9	7.90	7.76	97	104	8.71	9.39

Nd: None detected (less than the limit of detection of 0.05 mg/L)

ASV: Air saturation value (100% ASV = 9.09 mgO2/L at 20ºC)

The total hardness and alkalinity of the batch of softened Elendt M4 medium used were 168 and 39 mg/L as CaCO3, respectively

Continuous monitoring of an additional vessel containing dilution medium = 21.2 to 21.6°C

# Geometric mean between concentration of trace constituent at t=0 and t=48h

 

Table 2. Number of introduced daphnids and incidence of immobility in the definitive test

Nominal loading rate of the test substance	Measured tracer constituent #	Cumulative numbers of immobile Daphnia
		24 hours						48 hours
		R1	R2	R3	R4	Total	%	R1	R2	R3	R4	Total	%
Control	nd	0	0	0	0	0	0	0	0	0	0	0	0
1.94	1.26	0	0	0	0	0	0	0	0	0	0	0	0
4.27	2.40	0	0	0	0	0	0	0	0	1	3	4	20
9.39	5.71	0	0	0	1	1	5	3	4	0	2	9	45
20.7	10.2	4	2	1	4	11	55	3	4	5	5	17	85
45.5	23.6	3	1	5	4	13	65	1	5	5	5	16	80
100	46.3	5	5	5	5	20	100	5	5	5	5	20	100

nd: None detected (less than the limit of detection of 0.05 mg/L)

R: Replicate number

# Geometric mean between concentration of trace constituent at t=0 and t=48h

Validity criteria fulfilled:

yes

Conclusions:

The test substance 48h-EL50 was 10.9 mg/L (C.I. 7.98 - 14.80 mg/L) based on nominal loading rates.The NOELR was 1.94 mg/L.

Executive summary:

The acute toxicity to Daphnia magna was carried out according to OECD TG 202 Daphnia sp., Acute Immobilisation Test, EU Method C.2, US EPA 850.1010 and Japanese, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147 guidelines under GLP. Groups of twenty Daphnia, less than 24 hours old, were exposed to water accommodated fractions (WAF) of test substance prepared from aqueous mixtures with nominal loading rates of 1.94, 4.27, 9.39, 20.7, 45.5 and 100 mg/L. Aliquots of the test substance were added to softened Elendt M4 medium; these mixtures were stirred for approximately 24 hours in the dark and then left to stand for approximately 24 hours in the dark before the WAFs were removed and filtered (0.2 μm pore size), before being used directly as test media. The test substance comprised several constituents. One constituent was analysed as a tracer to assess the stability of the WAF system. The analysis described within this report was the analysis of one of the constituents measured at a specific wavelength using the gas chromatography with mass spectrometric detection system (GC-MS) method. In samples of freshly prepared media, the concentrations of the tracer constituent at the beginning of the test ranged between 1.44 to 48.4 mg/L. Measured levels of the tracer constituent in samples at the end of the test ranged between 1.11 and 44.3 mg/L, between 77 and 107 % of the initially measured concentrations. The geometric mean of the measured concentrations of the tracer constituent was 1.26, 2.40, 5.71, 10.2, 23.6 and 46.3 mg/L for the remaining levels. Since the test substance is a complex mixture, its toxicity cannot be attributed to a single constituent or a mixture of constituents, but to the test substance as a whole. Therefore the nominal loading rates of the test substance were used in the determination of study end-points, as it is recommended in the OECD Guidance Document No 23 on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD, 2000). Observations of the Daphnia in each control and test vessel were made after approximately 24 and 48 hours. After 48 hours, 100% immobilisation occurred at the nominal loading rate of 100 mg/L, and no immobilisation occurred at the nominal loading rate of 1.94 mg/L. The following study end-points were determined using the nominal loading rates of the test substance at 48 hours: EL50: 10.9 (C.I. 7.98 – 14.80) mg/L. The No Observed Effect Loading Rate (NOELR) was determined to be 1.94 mg/L.

Description of key information

EL50 (invertebrates) = 10.9 mg/L ; WAF loading 48hour-freshwater, OECD TG 202, 2015

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

10.9 mg/L

Additional information

The acute toxicity to Daphnia magna was carried out according to OECD TG 202 Daphnia sp., Acute Immobilisation Test, EU Method C.2, US EPA 850.1010 and Japanese, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147 guidelines under GLP. Groups of twenty Daphnia, less than 24 hours old, were exposed to water accommodated fractions (WAF) of test substance prepared from aqueous mixtures with nominal loading rates of 1.94, 4.27, 9.39, 20.7, 45.5 and 100 mg/L. Aliquots of the test substance were added to softened Elendt M4 medium; these mixtures were stirred for approximately 24 hours in the dark and then left to stand for approximately 24 hours in the dark before the WAFs were removed and filtered (0.2 μm pore size), before being used directly as test media. The test substance comprised several constituents. One constituent was analysed as a tracer to assess the stability of the WAF system. The analysis described within this report was the analysis of one of the constituents measured at a specific wavelength using the gas chromatography with mass spectrometric detection system (GC-MS) method. In samples of freshly prepared media, the concentrations of the tracer constituent at the beginning of the test ranged between 1.44 to 48.4 mg/L. Measured levels of the tracer constituent in samples at the end of the test ranged between 1.11 and 44.3 mg/L, between 77 and 107 % of the initially measured concentrations. The geometric mean of the measured concentrations of the tracer constituent was 1.26, 2.40, 5.71, 10.2, 23.6 and 46.3 mg/L for the remaining levels. Since the test substance is a complex mixture, its toxicity cannot be attributed to a single constituent or a mixture of constituents, but to the test substance as a whole. Therefore the nominal loading rates of the test substance were used in the determination of study end-points, as it is recommended in the OECD Guidance Document No 23 on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD, 2000). Observations of the Daphnia in each control and test vessel were made after approximately 24 and 48 hours. After 48 hours, 100% immobilisation occurred at the nominal loading rate of 100 mg/L, and no immobilisation occurred at the nominal loading rate of 1.94 mg/L. The following study end-points were determined using the nominal loading rates of the test substance at 48 hours: EL50: 10.9 (C.I. 7.98 – 14.80) mg/L. The No Observed Effect Loading Rate (NOELR) was determined to be 1.94 mg/L.