4-(2,6,6-trimethylcyclohex-2-ene-1-yl)-but-3-ene-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:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data of test chemical

Justification for type of information:

Data for the test chemical is from handbook or collection of data

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

WoE report is prepared based on short term toxicity to aquatic invertebrate study:
2, 3 and 4th

GLP compliance:

no

Test organisms (species):

Daphnia magna

Details on test organisms:

2. TEST ORGANISM
- Common name: Water flea
- Age of parental stock (mean and range, SD): < 24 hrs old

- Other: Juvenile daphnids (<24 hours old) produced from an in-house culture of adults were maintained at the contract laboratory under test conditions for 45 days. During the 48 hours prior to testing, the daphnid culture was maintained in 100% dilution water under static, renewal conditions for 48 hours. There was no mortality during the 48 hours prior to test and the test organisms appeared free of disease, injuries, or abnormalities. The daphnid culture produced young before day 12 and a subsample of adults produced on average, more than 3 young per day during the 7days prior to the beginning of the test. The test substance was provided via an intermittent flow proportional diluter.

Test type:

static

Water media type:

freshwater

Total exposure duration:

48 h

Hardness:

20 - 3.20 mmol/l

Test temperature:

20.0 - 22.7 ºC

pH:

7.3- 8.5

Dissolved oxygen:

2. 8.4-9.1 mg/L
3. 8.8 - 9.1 mg/l

Conductivity:

2. 490-620 umhos/cm
3. 550 - 650 µS/c

Nominal and measured concentrations:

1. 5 nominal concentrations 0.78, 1.3, 2.2, 3.6, and 6.0 mg/L were used for the toxicity testing

Details on test conditions:

1. - No. of organisms per vessel: 10
- No of replicates: 2
- Photoperiod: 16 hours of light and 8 hours of darkness

3. Alkalinity up to pH 4.3: 0.80 – 1.00 mmol/l
Molar ratio Ca:Mg: about 4 : 1
- Test vessel: Test tubes (glass), nominal volume: 20 ml
- Test volume : 10 mL
- Loading (animals/ml): 0.5
- Number of animals/vessel: 5
- Total number of animals/conc.: 20
- Number of replicates: 4
- Illumination: Artificial light, 16 light : 8 h dark
- Test parameter: Swimming ability of the test animalsafter 0, 24 and 48 h

Reference substance (positive control):

not specified

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.65 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 1st study

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

5.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 2nd study

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

3.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: confidence limits 95%:3.36 - 4.92 mg/l

Remarks:

3rd study

Reported statistics and error estimates:

1. Probit method
3. The EC50 (48 h) calculated using the Probit analysis

3 study:

The number of mobile daphnids after hours of exposure in the different test concentrations was as following:

Concentration [mg/l]	repl. 1		repl. 2		repl. 3		repl. 4		Sum
	24	48	24	48	24	48	24	48	24	48
0	5	5	5	5	5	5	5	5	20	20
1	5	5	5	5	5	5	5	5	20	20
2	5	4	5	5	5	5	5	4	20	18
4	4	4	4	4	4	1	4	4	16	13
8	1	0	1	0	1	0	1	0	6	0
16	0	0	0	0	0	0	0	0	0	0

 

The EC50 (48 h) calculated using the Probit analyses, was 4.03 mg/l (confidence limits 95%:3.36 - 4.92 mg/l).

Concentration control analyses performed by HPLC indicate that initial concentration was between 90 and 92 % of nominal

conentration and reduction during the test period (48 h) was below 20%. The percent recovery was as follow:

nominal conc. [mg/l]	measured (0h; %)	measured (48h; %)
1	91.1	78.2
2	90.9	76.9
4	92.0	74.8
8	91.4	73.8
16	90.1	75.4

Validity criteria fulfilled:

not specified

Conclusions:

The EC50 value of test chemical was observed to be in the range 2.6 to 5.7 mg/L when daphnia magna was exposed to for 48 hrs. on the basis of value it can be concluded that the test chemical is toxic to the aquatic environment.

Executive summary:

Data available for the test chemical was reviewed to determine its toxic nature upon the growth and mobility of aquatic invertebrate. The studies are as mentioned below:

The first study was to determine the effect of test chemical on the immobilisation of test organism daphnia magna. Test conducted in accordance with OECD guideline 202 (Daphnia sp. Acute Immobilisation Test). Analytically monitoring of the test sample conducted by using HPLC/UV detector. Juvenile daphnia (<24 hours old) produced from an in-house culture of adults were maintained at the contract laboratory under test conditions for 45 days. During the 48 hours prior to testing, the daphnid culture was maintained in 100% dilution water under static, renewal conditions for 48 hours. There was no mortality during the 48 hours prior to test and the test organisms appeared free of disease, injuries, or abnormalities. The daphnid culture produced young before day 12 and a subsample of adults produced on average, more than 3 young per day during the 7days prior to the beginning of the test. The test substance was provided via an intermittent flow proportional diluter. 5 nominal concentrations 0.78, 1.3, 2.2, 3.6, and 6.0 mg/L were used for the toxicity testing. 10 daphnia per vessel added and test conducted in duplicates. After the exposure of test chemical for 48 hrs, toxicity were measured on the basis of immobility and mortality rate of daphnia magna. The EC50 value was determine to be 2.65 mg/l and LC50 was observed at the 3.11 mg/l. Based on the above effect chemical consider to be toxic and classified as aquatic chronic 2 as per the CLP classification criteria.

Similarly in the second study a Daphnia immobilization static study was conducted following Guideline “Acute Toxicity for Daphnia” (C.2), Directive 92/69/EEC by providing the exposure period of 48 hrs. Test conducted under the static system. After the exposure of 48 hrs, effect on the daphnia magna were observed and measured on the basis of immobility. The EC50 was determine to be 5.7 mg/l.

Above studies was supported by the another third study. To study the effects of test chemical on aquatic invertebrates test was carried out according to the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). The 48-h LC50 value is based on measured data. A synthetic fresh water was used for culture and test purposes. Properties of this medium: Total hardness: 2,20 - 3,20 mmol/l, Alkalinity up to pH 4.3: 0,80 - 1,00 mmol/l, Molar ratio Ca:Mg: about 4 : 1, pH value: 7,5 - 8,5, Conductivity: 550 - 650 μS/cm. After preparation the M4 medium was aerated for approximately 24 h until saturation. The test substance was tested in the range of concentrations between 16 and 1 mg/l. The dilution factor was 2. The number of mobile daphnids after 24 and 48 hours of exposure in the different test concentrations was measured. The EC50 (48 h) calculated using the Probit analyses The EC 50 value of test chemical was observed to be 3.7 mg/L when daphnia magna was exposed to for 48 hrs. On the basis of this value it can be concluded that the test chemical is toxic to the aquatic environment and can be considered as Aquatic acute 2 as per the classification criteria for aquatic environment.

Thus based on the overall studies, chemical consider to be toxic and can be consider to be classified as aquatic chronic 2 as per the CLP classification criteria.

Description of key information

After the exposure of test chemical for 48 hrs, toxicity were measured on the basis of immobility and mortality rate of daphnia magna. The EC50 value was determine to be 2.65 mg/l and LC50 was observed at the 3.11 mg/l. Based on the above effect chemical consider to be toxic and classified as aquatic chronic 2

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

2.65 mg/L

Additional information

Data available for the test chemical was reviewed to determine its toxic nature upon the growth and mobility of aquatic invertebrate based on the available study of structurally similar read across substances. The studies are as mentioned below:

 

The first study was to determine the effect of test chemical on the immobilisation of test organism daphnia magna. Test conducted in accordance with OECD guideline 202 (Daphnia sp. Acute Immobilisation Test). Analytically monitoring of the test sample conducted by using HPLC/UV detector. Juvenile daphnia (<24 hours old) produced from an in-house culture of adults were maintained at the contract laboratory under test conditions for 45 days. During the 48 hours prior to testing, the daphnid culture was maintained in 100% dilution water under static, renewal conditions for 48 hours. There was no mortality during the 48 hours prior to test and the test organisms appeared free of disease, injuries, or abnormalities. The daphnid culture produced young before day 12 and a subsample of adults produced on average, more than 3 young per day during the 7days prior to the beginning of the test. The test substance was provided via an intermittent flow proportional diluter. 5 nominal concentrations 0.78, 1.3, 2.2, 3.6, and 6.0 mg/L were used for the toxicity testing. 10 daphnia per vessel added and test conducted in duplicates. After the exposure of test chemical for 48 hrs, toxicity were measured on the basis of immobility and mortality rate of daphnia magna. The EC50 value was determine to be 2.65 mg/l and LC50 was observed at the 3.11 mg/l. Based on the above effect chemical consider to be toxic and classified as aquatic chronic 2 as per the CLP classification criteria.

 

Similarly in the second study a Daphnia immobilization static study was conducted following Guideline “Acute Toxicity for Daphnia” (C.2), Directive 92/69/EEC by providing the exposure period of 48 hrs. Test conducted under the static system. After the exposure of 48 hrs, effect on the daphnia magna were observed and measured on the basis of immobility. The EC50 was determine to be 5.7 mg/l.

 

Above studies was supported by another third study. To study the effects of test chemical on aquatic invertebrates test was carried out according to the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). The 48-h LC50 value is based on measured data. A synthetic fresh water was used for culture and test purposes. Properties of this medium: Total hardness: 2,20 - 3,20 mmol/l, Alkalinity up to pH 4.3: 0,80 - 1,00 mmol/l, Molar ratio Ca:Mg: about 4 : 1, pH value: 7,5 - 8,5, Conductivity: 550 - 650 μS/cm. After preparation the M4 medium was aerated for approximately 24 h until saturation. The test substance was tested in the range of concentrations between 16 and 1 mg/l. The dilution factor was 2. The number of mobile daphnid after 24 and 48 hours of exposure in the different test concentrations was measured. The EC50 (48 h) calculated using the Probit analyses The EC 50 value of test chemical was observed to be 3.7 mg/L when daphnia magna was exposed to for 48 hrs. On the basis of this value it can be concluded that the test chemical is toxic to the aquatic environment and can be considered as Aquatic chronic 2 as per the classification criteria for aquatic environment.

 

Thus based on the overall studies, chemical consider to be toxic and can be consider to be classified as aquatic chronic 2 as per the CLP classification criteria.