Reaction mass of 1,2,3,4,4a,5,6,7-octahydro-2,5,5-trimethyl-2-naphthol and 4-(2,6,6-trimethyl-1-cyclohexen-1-yl)butan-2-one and 4-(2,6,6-trimethyl-2-cyclohexen-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:

17 Nov 2017 - 19 Dec 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:

April 13, 2004

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:

2000

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: WAFs prepared at individual loading rates of 10, 18, 32, 56 and 100 mg/L.
- Sampling method: Samples for possible analysis were taken from all test concentrations and the control at t=0 h and t=48 h. 4.0 mL was taken from the approximate centre of the test vessels.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
- At the end of the exposure period, the replicates were not pooled at each concentration before sampling. Instead, samples were taken from one replicate per concentration for all test concentrations.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Preparation of test solutions started with loading rates individually prepared ranging between 1.0 and 100 mg/L. A three-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in medium. The obtained mixtures were allowed to settle for a period of one hour. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning and used as test concentrations. All test solutions were clear and colorless at the end of the preparation procedure.
- Controls: Test medium without test item or other additives

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus 1820.
- Source: In-house laboratory culture with a known history
- Age of parental stock: > 2 weeks
- Age at test initiation: <24 h
- Feeding during test: no

BREEDING
- Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel.
- Maximum age of the cultures: 4 weeks
- Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
- Temperature of medium: 18-22°C
- Feeding: Daily, a suspension of fresh water algae.
- Culture medium: M7

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

180 mg/L CaCO3

Test temperature:

19 - 20°C

pH:

At t=0 h: 7.9
At t=48 h: 8.1-8.2

Dissolved oxygen:

At t=0 h: 8.4-9.1 mg/L
At t=48 h: 7.8-8.3 mg/L

Nominal and measured concentrations:

Nominal: WAFs prepared at individual loading rates of 10, 18, 32, 56 and 100 mg/L.
Measured: At t=0 h: 9.6, 12, 13, 38 and 48 mg/L. The measured concentration remained stable throughout the test, i.e. were 95 - 106% relative to initial. Based on these results, effect parameters were expressed as measured initial concentrations. See Table 3 in 'Any other information on results' for measured concentrations for all samples throughout the test.

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 mL, all-glass, airtight closed with no headspace
- Aeration: no
- No. of organisms per vessel: 5 daphnids
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO medium, prepared with tap water purified by Reverse Osmosis
- Culture medium different from test medium: yes
- Intervals of water quality measurement: pH and dissolved oxygen: at the beginning and at the end of the test, for all concentrations and the control. Temperature of medium: continuously in a temperature control vessel, beginning at the start of the test.

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

EFFECT PARAMETERS MEASURED
- Immobility (including mortality): at 24 hours and at 48 hours.

RANGE-FINDING STUDY
- Test concentrations: Control, WAFs prepared individually at 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 (performed Jan 2018)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

11 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Remarks on result:

other: 95% confidence interval: 8.7-13 mg/L

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

< 96 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

9.6 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Details on results:

No biologically significant immobility was observed in the control throughout the exposure period. After 24 hours of exposure, all daphnids were immobilized in the measured concentrations of 38 and 48 mg/L. At the end of the test, a dose-related increase of immobility was observed at all test concentrations.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: The 48h-EC50 was 0.54 mg/L with a 95% confidence interval between 0.49 and 0.61 mg/L. The responses in this reference test with K2Cr2O7 are within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was between 0.3 and 1.0 mg/L. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at Charles River Den Bosch.

Reported statistics and error estimates:

48h-EC50-value was calculated from the weibits of the percentages of affected daphnids and the logarithms of the corresponding test item concentrations (initially measured) using the maximum likelihood estimation method. ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Table 2: Number of Introduced Daphnids and Incidence of Immobility in the Final Test

Time (h)	Replicate	POLYAMBROL; Measured concentration (mg/L)
		Control	9.6	12	13	38	48
0	A	5	5	5	5	5	5
	B	5	5	5	5	5	5
	C	5	5	5	5	5	5
	D	5	5	5	5	5	5
	Total introduced	20	20	20	20	20	20
24	A	0	0(5)	0(2)	0	5	5(4)
	B	0	0(3)	0(3)	0	5	5
	C	0	1	0	0	5	5
	D	0	0	0	0	5	5
	Total immobilised	0	1	0	0	20	20
	Effect %	0	5	0	0	100	100
48	A	0	0(1)	1	4	5	5
	B	0(1)	3^#	3	3	5	5
	C	0	2	2	5	5	5
	D	0	2	3	5	5	5
	Total immobilised	0	7	9	17	20	20
	Effect %	0	35	45	85	100	100

( ) between brackets: number of daphnids observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.

^#Microscopic observation revealed no test item attached to the daphnids.

Table 3: Final Test: Test Samples

Time of sampling [hours]	Date of sampling	Date of analysis^1	Loading rate^2 [mg/L]	Concentration analyzed [mg/L]	Relative to initial [%]
0	11 Dec 2017	18 Dec 2017	0	n.d.
			10	9.55
			18	11.8
			32	12.9
			56	37.9
			100	48.2
48	13 Dec 2017	18 Dec 2017	0	n.d.	n.a.
			10	9.10	95
			18	11.4	97
			32	13.1	102
			56	39.0	103
			100	50.9	106

¹     Samples were stored in the freezer (≤ -15°C) until the day of analysis.

²     Awater accommodated fraction (WAF)prepared at the loading rate.

n.d. Not detected.

n.a. Not applicable

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks' for details on validity criteria.

Conclusions:

The 48h-EC50 for Daphnia magna exposed to POLYAMBROL was 11 mg/L based on measured concentrations (95% confidence interval between 8.7 and 13 mg/L).

Executive summary:

In a 48 h acute toxicity test conducted according to OECD guideline 202, Daphnia magna were exposed to Water Accommodated Fractions of the test item individually prepared at 10, 18, 32, 56 and 100 mg/L. Initial measured concentrations were 9.6, 12, 13, 38 and 48 mg/L, and remained stable throughout the test duration (i.e. 95 -106% relative to initial). Therefore, effect parameters were expressed as initial measured concentrations. No biologically significant immobility or other effects were observed in the control. After 24 h of exposure, all daphnids were immobilized in the measured concentrations of 38 and 48 mg/L. At the end of the test, a dose-related increase of immobility was observed at measured concentrations of 9.6 mg/L and higher. The 48h-EC50 for immobility was calculated to be 11 mg/L (95% confidence interval 8.7 -13 mg/L), the NOEC was < 9.6 mg/L, and the LOEC was 9.6 mg/L. The study is considered to be reliable without restrictions.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

11 mg/L