Reaction mass of 1-(2,6,6-trimethylcyclohex-2-en-1-yl)hepta-1,6-dien-3-one and 1-(2,6,6-trimethylcyclohex-1-en-1-yl)hepta-1,6-dien-3-one

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: read-across from a guideline study

Justification for type of information:

The acute fish toxicity of Hexalon is based on read across from Galbascone. The documentation is presented in the Endpoint summary of Aquatic toxicity.

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

4.1 mg/L

Basis for effect:

mortality (fish)

Remarks on result:

other: read-across from Galbascone

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 August 2015 - 15 November 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Information is used for read across to Hexalon

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

(1992)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

(2008)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23, December 14, 2000

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Test concentrations were verified by chemical analysis. Samples for analysis were taken from all test concentrations with surviving fish and the control according to the schedule below:

Frequency At the start and the end of all 24-hour renewal periods.
Volume 3.0 mL from the approximate centre of the test vessels
Storage Samples were stored in a freezer until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The batch of GALBASCONE tested was a clear colourless liquid and not completely soluble in test medium at loading rates above 10 mg/L. No correction was made for the composition of the test item.

All test solutions were prepared separately applying one day of magnetic stirring in a closed vessel followed by a stabilization period of one day in order to reach the maximum solubility of the test item in the test medium. The resulting clear and colourless solutions (concentrations up to and including 10 mg/L) and the aqueous mixtures (concentrations above 10 mg/L) were siphoned off. The final test solutions were all referred to as Water Accommodated Fractions (WAFs).

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM
- Common name: Carp (Cyprinus carpio)
- Source: Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, The Netherlands
- Age at study initiation: not reported; F1 from a single parent-pair bred in UV-treated water
- Length at study initiation (final test, mean): 2.2 ± 0.2 cm
- Weight at study initiation (final test, mean): 0.38 ± 0.13 g
- Method of breeding: F1 from a single parent-pair bred in UV-treated water.
- Feeding during test: No feeding from 24 hours prior to the test and during the total test period.

ACCLIMATION
- Acclimation period: at least 12 days after delivery
- Acclimation conditions: same as test, i.e. adjusted ISO medium
- Type of food: pelleted fish food (Cyprico Crumble Excellent (300-500 µm))
- Feeding frequency: daily
- Health during acclimation: <5% mortality observed during the seven days prior to the start of the test

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

180 mg CaCO3/L

Test temperature:

21.0 - 21.7°C

pH:

7.0 - 7.9

Dissolved oxygen:

fresh solutions: 8.6-9.3 mgO2/L
used solutions: 3.0-6.4 mgO2/L

Nominal and measured concentrations:

Based on the results of the range-finding test (biological effects observed between 1.0 and 10 mg/L) the following test concentrations were assigned to the final test: WAFs prepared at loading rates of 1.0, 2.2, 4.6, 10 and 22 mg/L.

Measured test concentrations: see Tables 1 and 2 in field 'Any other information on results'

The effect concentrations are based on the arithmetic mean of geometric mean measured concentrations since the average measured concentrations derived from the 24 h intervals decreased to 44-71% of initial at the end of the various 24-hour renewal periods.

Details on test conditions:

TEST SYSTEM
- Test vessel: 3.2 litres, all-glass
- Type: closed (air-tight)
- Fill volume: 3.2 litres of test solution
- Aeration: no
- Renewal rate of test solution: 24 h
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.83 g fish/litre, i.e.7 fish per 3.2 litres of test medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO medium with a hardness of 180 mg CaCO3 per litre and a pH of 7.7 ± 0.3

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

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Mortality (after 3.5, 24, 48, 72 and 96 hours following the start of exposure)
- Other effects: dead or severely distressed fish - daily

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.1- 2.2

- Range finding study
- Test concentrations: WAFs prepared at loading rates of 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

pentachlorophenol (January 2015)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL: 3.3-7.7 mg/L (based on the arithmetic mean of the geometric mean measured concentrations)

Details on results:

The effect, i.e. 50% mortality, was reached already after 48 hours of exposure.

No mortality was observed in the control throughout the test.

pH and temperature remained within the recommended guideline ranges (pH: 6.0-8.5, constant within 1 unit; temperature 20-24°C, constant within 2°C).

The oxygen concentrations remained within the recommended guideline ranges (oxygen > 60% of air saturation), except for the old medium of the 10 mg/L test item concentration on day 1. In that test solution the oxygen concentration had decreased to 3.0 mg/L. Since the test item was treated as volatile the test solutions could not be aerated. Based on experience, fish are not affected by temporarily lower oxygen concentrations. Therefore, the fish mortality observed after 48 hours of exposure cannot be directly related to this temporarily lower oxygen concentration. Moreover, fish mortality was expected at this concentration level, based on the results of the range-finding test where the oxygen concentrations were at least 60% of air saturation at all times.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Mortality: 100% at 0.46 mg/L
- LC50: 0.32 mg/L
- Other: effect (i.e. 50% mortality) reached already within 48 h of exposure

Reported statistics and error estimates:

LC50 determination
The LC50 could not be determined using the maximum likelihood estimation method with the probits of the percentages of dead fish as function of the logarithms of the corresponding concentrations. This was because there was no concentration between the highest concentration (A) at which 0% mortality and the lowest concentration (B) at which 100% mortality occurred. Instead, the LC50 was calculated as (AB)^½, with A and B being limits of the 95% confidence interval.

Sublethal observations / clinical signs:

Range-finding test (static)

All fish exposed to the highest test concentration died within 4 hours of exposure. The actual test concentration was 45 mg/L at the start of the test and this concentration remained stable during the 4 hours of exposure (95% of initial).

Two out of three fish exposed to the middle test concentration also died within 4 hours of exposure. The actual test concentration was 8.7 mg/L at the start of the test and this concentration remained stable for at least 28 hours (85% of initial). The surviving fish after the 96 -hour test period was observed to be discoloured and stopped swimming from time to time. At the end of the test period the concentration had decreased to 62% of initial.

No mortality or clinical effects were observed in the lowest test concentration during the test period. The actual test concentration was 1.2 mg/L at the start of the test and this concentration decreased to 24% of initial at the end of the test.

The expected LC₅₀was between concentrations obtained in WAFs prepared at loading rates of 1.0 and 10 mg/L.

Table 1: Concentrations of the test item in test medium - final test

Time of sampling [hours]	Loading rate^1 [mg/L]	Concentration analysed [mg/L]	Relative to initial [%]
0	0	n.d.
	1.0	1.12
	2.2	2.25
	4.6	4.27
	10	9.16
	22	21.0
24 (Old)	0	n.d.
	1.0	0.563	50
	2.2	0.995	44
	4.6	2.52	59
	10	5.45	59
24 (Fresh)	0	n.d.
	1.0	1.10
	2.2	2.09
	4.6	4.45
	10	10.4
48 (Old)	0	n.d.
	1.0	0.596	54
	2.2	1.49	71
	4.6	2.77	62
	10	6.78	65
48 (Fresh)	0	n.d.
	1.0	1.14
	2.2	1.96
	4.6	4.22
	10	8.64
72 (Old)	0	n.d.
	1.0	0.572	50
	2.2	1.19	61
	4.6	2.86	68
72 (Fresh)	0	n.d.
	1.0	0.979
	2.2	1.89
	4.6	3.72
96 (Old)	0	n.d.
	1.0	0.587	60
	2.2	1.14	60
	4.6	2.63	71

¹          A water accommodated fraction (WAF) prepared at the loading rate.

n.d.    Not detected.

Table 2: Mean measured concentrations

GALBASCONE WAF prepared at x mg/L	Geometric mean concentration (mg/L)				Mean concentration 0-96 h (mg/L)
	t=0-24 h	t=24-48 h	t=48-72 h	t=72-96 h
1.0	0.79	0.81	0.81	0.76	0.79
2.2	1.5	1.8	1.5	1.5	1.6
4.6	3.3	3.5	3.5	3.1	3.3
10	7.1	8.4			7.7
22	21^1				21^1

¹Initial concentration

Table 3: Incidence of mortality and total mortality during the final test

Mean conc. GALBASCONE (mg/L)	Initial number of fish	Cumulative mortality					Total Mortality (%)
		3½h	24h	48h	72h	96h
control	7	0	0	0	0	0	0
0.79	7	0	0	0	0	0	0
1.6	7	0	0	0	0	0	0
3.3	7	0	0	0	0	0	0
7.7	7	0	0	7	7	7	100
21	7	7	7	7	7	7	100

Table 4: Clinical effects observed during the final test

Mean conc. GALBASCONE (mg/L)	Time of recording (hours)	Specification of effects	Relative number
Control, 0.79, 1.6   3.3           7.7	0-96   3½ 24   48-96     3½   24	No abnormalities   Discoloured and hypoactive Snapping at the surface No abnormalities Discoloured No abnormalities   Immobile Loss of equilibrium Immobile	7/7   7/7 6/7 1/7 1/7 6/7   4/7 3/7 7/7

Validity criteria fulfilled:

yes

Remarks:

no mortality in controls; test conditions were maintained constant throughout the test; for dissolved oxygen criterion and concentrations please refer to 'Overall remarks'

Conclusions:

The 96 h LC50 value in fish (Cyprinus carpio) is 5.0 mg/L.

Executive summary:

A study was performed to assess the acute toxicity of the substance to fish (Cyprinus carpio). The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 203 and GLP. In a semi-static test set-up in air-tight closed 3.2-L vessels, seven fish per concentration were exposed to 5 concentrations of the substance in test water (water accommodated fractions prepared separately at loading rates of 1.0, 2.2, 4.6, 10 and 22 mg/L) and a control. Throughout the test, test solutions were refreshed in intervals of 24 hours. After 3.5 hours all fish in the highest treatment group, and after 48 hours all fish in the second highest treatment group had died. Clinical effects (such as discolouration, hypoactive swimming, loss of equilibrium or immobility) and incidences of mortality were recorded for the control and each test group at all timepoints and the following effect value was determined: 96-h LC50 = 5.0 mg/L.

All results are expressed in terms of mean measured concentrations. Measured concentrations were in agreement with initial loading rates in fresh solutions (81 -114%) but decreased to 44 -71 % of initial during the refreshment periods of 24 hours (in used solutions). Therefore, geometric mean measured concentrations were calculated for each time interval and concentration, and these values were used to calculate the 0 -96 h (arithmetic) mean measured concentrations.

Description of key information

The short-term toxicity to fish is assessed based on read-across from the close structural analogue Galbascone (CAS# 56973 -85 -4) and the LC50 value determined at 4.1 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

4.1 mg/L

Additional information

The information for Hexalon is based on read across from Galbascone (CAS# 56973 -85 -4). The read across justification is presented in the Endpoint summary of Aquatic toxicity. The executive summary of the acute fish test of Galbascone is presented in the present section.

Galbascone and its acute fish toxicity

For Galbascone, a study was performed to assess the acute toxicity of the substance to fish (Cyprinus carpio). The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 203 and GLP. In a semi-static test set-up in air-tight closed 3.2-L vessels, seven fish per concentration were exposed to 5 concentrations of the substance in test water (water accommodated fractions prepared separately at loading rates of 1.0, 2.2, 4.6, 10 and 22 mg/L) and a control. Throughout the test, test solutions were refreshed in intervals of 24 hours. After 3.5 hours all fish in the highest treatment group, and after 48 hours all fish in the second highest treatment group had died. Clinical effects (such as discolouration, hypoactive swimming, loss of equilibrium or immobility) and incidences of mortality were recorded for the control and each test group at all timepoints and the following effect value was determined: 96-h LC50 = 5.0 mg/L. All results are expressed in terms of mean measured concentrations. Measured concentrations were in agreement with initial loading rates in fresh solutions (81 -114%) but decreased to 44 -71 % of initial during the refreshment periods of 24 hours (in used solutions). Therefore, geometric mean measured concentrations were calculated for each time interval and concentration, and these values were used to calculate the 0-96 h (arithmetic) mean measured concentrations.