Reaction mass of 4-(2,6,6-trimethylcyclohex-2-ene-1-yl)-but-3-ene-2-one and 4-(2,6,6-trimethylcyclohex-1-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:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Experimental test result performed using standard OECD test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

GLP compliance:

no

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

The test solution was prepared by dissolving 200mg of test chemical in 200ml of M7 medium to get the final concentration of 1000 mg/L. Stock solution was verified analytically by UV-Vis Spectrophotometer. The final solubility value obtained after analytical detection was 62.423 mg/L. Further, exposure concentrations of 0, 0.987, 1.481, 2.222, 3.333 and 5 mg/l, respectively was from the stock solution.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain/clone: Eggs of Daphnia magna were obtained from MicroBio tests Kleimoer 15B-9030 MARIAKERKE (GENT) BELGIUM
- Feeding during test: no
- Other: A population of parthenogenetic females of synchronized age structure has been maintained for more than 2 years in the test facility under constant temperature conditions (18 to 22°C) at a 16 : 8 hour light-dark photoperiod (illumination: < 1000 lux). The culture media (M7 medium') was partly renewed twice a week. During the maintenance of test organism, test daphnids were fed with unicellular green algae (Selenestrum capricornutum).

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

> 140 mg of CaCO3

Test temperature:

25°C

pH:

7.3-7.9

Dissolved oxygen:

5.0-7.5

Nominal and measured concentrations:

Nominal concentrations: 0, 0.987, 1.481, 2.222, 3.333 and 5 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beaker
- Aeration: No aeration during experiment
- Material, size, headspace, fill volume: 25 ml of glass beaker filled with 20 ml media having headspace of 5 ml
- No. of organisms per vessel: 20 daphnids

OTHER TEST CONDITIONS
- Photoperiod:16 hours light and 8 hours dark
- Light intensity: 1000 – 1500 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The criterion of immobilisation used in this study was the absence of response to physical stimulation. In addition to observations on mortality at 24, and 48 hours, type and incidence of sub-effective effects compared with control Daphnia were observed.

Reference substance (positive control):

yes

Remarks:

potassium dichromate was used as a reference substance for the study.

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Results with reference substance (positive control):

The 24 hr EC50 value of reference substance potassium dichromate was determined to be 0.831 mg/l.

Table: Assessment of test concentrations

Sr. no.	Concentrations (mg/l)	Wavelength (nm)	Absorbance	Temperature (°C)
1	blank	0.00	0.00	25
2	0.500	231	0.0496	25
3	1.000	231	0.0948	25
4	1.500	231	0.1291	25
5	2.000	231	0.1660	25
6	2.500	231	0.2091	25
7	3.000	231	0.2450	25
8	3.500	231	0.2907	25
9	4.000	231	0.3405	25
10	4.500	231	0.3902	25
11	5.00	231	0.4315	25

 

The absorbance and concentrations were recorded at 231 nm.

Table: Concentration after analytical Determination

Sr. No	Concentrations (mg/L)	Analytical Concentrations (0 hour)	Analytical Concentrations (48 hour)
1	blank	0.00	0.00
2	5	4.887	4.032

 

OBSERVATIONS

 

IMMOBILIZATION AND INHIBITION

 

Sr. no.	Test concentrations(mg/L)	0 Hour	24 Hour	48 Hour	Cumulative Immobilization	Percent Inhibition
1	Control	-	-	-	-	-
2	0.987	-	-	-	-	-
3	1.481	-	-	-	-	-
4	2.222	-	-	-	-	-
5	3.333	-	1	-	1	10
6	5	-	2	-	2	20
		No abnormality observed	Immobility observed and slow mobility as compared to control	No immobility observed

 

  

pH, DO AND TEMPERATURE

Test Concentration(mg/L)	pH			Dissolved oxygen			Temperature °C
	0 Hour	24 Hour	48 Hour	0 Hour	24 Hour	48 Hour	0 Hour	24 Hour	48 Hour
Control	7.5	7.5	7.6	7.5	7.1	6.4	20.7	25.2	20.4
0.987	7.3	7.4	7.7	7.2	7.0	5.8	20.7	25.1	20.1
1.481	7.3	7.5	7.7	7.2	6.6	5.5	20.7	25.1	20.4
2.222	7.4	7.4	7.8	7.2	6.9	5.1	20.5	25.0	20.2
3.333	7.4	7.5	7.8	7.1	7.0	5.0	20.4	25.0	20.3
5	7.3	7.6	7.9	7.1	6.8	4.8	20.2	25.0	20.4

Validity criteria fulfilled:

yes

Remarks:

1. In the control, including the control containing the solubilising agent, not more that 10 percent of the daphnids should have been immobilized. 2. The dissolved oxygen concentration at the end of the test should be 3 mg/l in control and test vessels

Conclusions:

Based on nominal concentrations, experimental median effective concentrations [EC50 (48 h)] for test chemical on Daphnia magna was determined to be > 5.0 mg/L.

Executive summary:

An acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical on Daphnia magna. The test was performed in accordance to OECD guideline No. 202“Daphnia sp.,Acute Immobilization Test”. The test solution was prepared by dissolving 200mg of test chemical in 200ml of M7 medium to get the final concentration of 1000 mg/L. Stock solution was verified analytically by UV-Vis Spectrophotometer. The final solubility value obtained after analytical detection was 62.423 mg/L. Further, exposure concentrations of 0, 0.987, 1.481, 2.222, 3.333 and 5 mg/l, respectively was from the stock solution. Study was performed using 20 daphnids in a static system. Total 20 Daphnids/conc. were exposed to test chemical in 25 ml beakers in a volume of 20 ml of liquid solution containing both the chemical and media. The beakers were placed in a room at a temperature of 18 -22°C, hardness of water > 140 mg of CaCO3 and under a photoperiod of 16:8 hr light: dark conditions with light intensity 1000 – 1500 Lux, respectively. One control vessel was also run simultaneously during the study. The animals in control and test chemical concentrations were exposed for a period of 48 hour. Potassium dichromate was used as a reference substance for the study. The 24 hr EC50 value of reference substance was determined to be 0.831 mg/l. No Immobility were found in the control test animals and the dissolved oxygen concentration at the end of the test in the control and test vessel was ≥ 3 mg/l, thus validity criterion of the study has been fulfilled. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of effect of test chemical on mobility of the test organism, the median effect concentration (EC50 (48 h)) value was determined to be > 5 mg/L. Since the test chemical is readily biodegradable in water, test chemical was considered as non-toxic to aquatic invertebrates at environmental relevant concentrations and hence, considered to be 'not classified' as per CLP classification criteria.

Description of key information

An acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical on Daphnia magna (Experimental study report, 2019). The test was performed in accordance to OECD guideline No. 202“Daphnia sp.,Acute Immobilization Test”. The test solution was prepared by dissolving 200mg of test chemical in 200ml of M7 medium to get the final concentration of 1000 mg/L. Stock solution was verified analytically by UV-Vis Spectrophotometer. The final solubility value obtained after analytical detection was 62.423 mg/L. Further, exposure concentrations of 0, 0.987, 1.481, 2.222, 3.333 and 5 mg/l, respectively was from the stock solution. Study was performed using 10 daphnids in a static system. Total 10 Daphnids/conc. were exposed to test chemical in 25 ml beakers in a volume of 20 ml of liquid solution containing both the chemical and media. The beakers were placed in a room at a temperature of 18 -22°C, hardness of water > 140 mg of CaCO3 and under a photoperiod of 16:8 hr light: dark conditions with light intensity 1000 – 1500 Lux, respectively. One control vessel was also run simultaneously during the study. The animals in control and test chemical concentrations were exposed for a period of 48 hour. Potassium dichromate was used as a reference substance for the study. The 24 hr EC50 value of reference substance was determined to be 0.831 mg/l. No Immobility were found in the control test animals and the dissolved oxygen concentration at the end of the test in the control and test vessel was ≥ 3 mg/l, thus validity criterion of the study has been fulfilled. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of effect of test chemical on mobility of the test organism, the median effect concentration (EC50 (48 h)) value was determined to be > 5 mg/L. Since the test chemical is readily biodegradable in water, test chemical was considered as non-toxic to aquatic invertebrates at environmental relevant concentrations and hence, considered to be 'not classified' as per CLP classification criteria.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

5 mg/L

Additional information

Various experimental studies of the test chemical and supporting weight of evidence study for its structurally and functionally similar read across chemical were reviewed for short term toxicity to aquatic invertebrate end point which are summarized as below:

 

In an experimental study from study report (2019),an acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical on Daphnia magna. The test was performed in accordance to OECD guideline No. 202“Daphnia sp.,Acute Immobilization Test”. The test solution was prepared by dissolving 200mg of test chemical in 200ml of M7 medium to get the final concentration of 1000 mg/L. Stock solution was verified analytically by UV-Vis Spectrophotometer. The final solubility value obtained after analytical detection was 62.423 mg/L. Further, exposure concentrations of 0, 0.987, 1.481, 2.222, 3.333 and 5 mg/l, respectively was from the stock solution. Study was performed using 20 daphnids in a static system. Total 20 Daphnids/conc. were exposed to test chemical in 25 ml beakers in a volume of 20 ml of liquid solution containing both the chemical and media. The beakers were placed in a room at a temperature of 18 -22°C, hardness of water > 140 mg of CaCO3 and under a photoperiod of 16:8 hr light: dark conditions with light intensity 1000 – 1500 Lux, respectively. One control vessel was also run simultaneously during the study. The animals in control and test chemical concentrations were exposed for a period of 48 hour. Potassium dichromate was used as a reference substance for the study. The 24 hr EC50 value of reference substance was determined to be 0.831 mg/l. No Immobility were found in the control test animals and the dissolved oxygen concentration at the end of the test in the control and test vessel was ≥ 3 mg/l, thus validity criterion of the study has been fulfilled. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of effect of test chemical on mobility of the test organism, the median effect concentration (EC50 (48 h)) value was determined to be > 5 mg/L. Since the test chemical is readily biodegradable in water, test chemical was considered as non-toxic to aquatic invertebrates at environmental relevant concentrations and hence, considered to be 'not classified' as per CLP classification criteria.

 

Another acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical on Daphnia magna (A.M. Api, et.al, 2016). The test was performed in accordance to EU Method C.2 (Acute Toxicity for Daphnia), Directive 92/69/EEC in a static system. On the basis of the effect of test chemical on mobility of the test organism Daphnia magna, the 48hr median effect concentration (EC50) value was determined to be 5.7 mg/l. Since the test chemical is readily biodegradable in water, test chemical was considered as non-toxic to aquatic invertebrates and hence, considered to be 'not classified' as per CLP classification criteria.

 

In a supporting weight of evidence study, short term toxicity study was conducted to assess the effect of test chemical on the immobilisation of test organism daphnia magna (authoritative database, 2018 and secondary source, 2018). Test was performed in accordance with OECD guideline 202 (Daphnia sp. Acute Immobilisation Test). Analytically monitoring of the test sample was carried out by using HPLC/UV detector. Test organism, 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. Test chemical concentrations used for the study were 0.78, 1.3, 2.2, 3.6, and 6.0 mg/L (nominal concentrations) and 0.621, 1.14, 1.82, 2.99, and 5.32 (mean measured concentration), respectively. 10 daphnia per vessel added and tests were conductedl 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. Based on these effects, the 48 hr NOEC, EC50 and LC50 value was determine to be 1.14 mg/l, 2.65 mg/l and 3.11 mg/l, respectively. The measured concentrations after 24 and 48 hours were 80-89% (which is within the range of ± 20%) of the nominal concentrations, with the concentration being held steady throughout the test period. Therefore, the analysis of the results was based on nominal concentration. On the basis of result, test chemical was considered as toxic to aquatic invertebrates. Since the test chemical is readily biodegradable in water, test chemical was considered as non-toxic to aquatic invertebrates and hence, considered to be 'not classified' as per CLP classification criteria.

 

For the test chemical from secondary source (2004), short term toxicty to aquatic invertebrates test was carried out following the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). Daphnia magna (Water flea) of 2 -24 hr old was used as a test organism. Age of the stock animals was 2 -4 weeks old. Test organism was not fed during the study. Nominal test chemical concentration used for the study were 0, 1, 2, 4, 8 and 16 mg/l, respectively. The dilution factor was 2. Test chemical concentrations were verified analytically by HPLC. Study was performed using daphnids in a static system. Total 20 Daphnids/conc. were exposed to test chemical in 20 ml test vessel in a volume of 10 ml of liquid solution containing both the chemical and media. The beakers were placed in a room at a temperature of 18 -22°C, pH 7.5 to 8.5, DO of 8.8 to 9.1 mg/l, hardness of water 2.20 - 3.20 mM/l and under a photoperiod of 16:8 hr light: dark conditions with light intensity 550 – 650 µS/cm, respectively. One control vessel was also run simultaneously during the study. After a period of 0, 24 and 48 hrs, number of mobile daphnids were measured. The EC50 (48 h) was calculated using the Probit analyses. On the basis of the effect of test chemical on mobility of the test organism Daphnia magna, the 48hr median effect concentration (EC50) value was determined to be 3.7 mg/l (initial measured conc.). Since the test chemical is readily biodegradable in water, test chemical was considered as non-toxic to aquatic invertebrates and hence, considered to be 'not classified' as per CLP classification criteria.

 

On the basis of the overall results, it can be concluded that the test chemicalwas considered as toxic to aquatic invertebrates. Since, the test chemical is readily biodegradable in water, chemical was considered as non-toxic to aquatic invertebrates at environmental concentrations and hence, considered to be'not classified' as per CLP classification criteria.