2,2'-(2-methylpropylidene)bis[4,6-xylenol]

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:

24 June 2016 to 18 August 2016

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:

OECD guidelines for Testing of Chemicals, guideline No. 202: "Daphnia sp., Acute Immobilisation Test", Adopted April 13, 2004.

Deviations:

yes

Remarks:

See "Any other information" for details

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

Commission Regulation (EC) No 440/2008 of 30 May 2008, Part C: Methods for the determination of ecotoxicity, Publication No. L142, C.2. "Daphnia Sp. Acute Immobilisation Test".

Deviations:

yes

Remarks:

see "Any other information" for details

GLP compliance:

yes

Specific details on test material used for the study:

Identification: Lowinox® 22IB46
Appearance: White to cream coloured powder
Batch: WB44L0016
Purity/Composition: 99.7%
Test item storage: At room temperature
Stable under storage conditions until: 18 November 2018 (retest date)
Test item: 207366/A
Purity/composition correction factor: No correction factor required
Chemical name (IUPAC), synonym or trade name: 2,2’-(2-methylpropylidene)bis[4,6-xylenol]
CAS Number: 33145-10-7
Highly reactive to water: Not indicated
Highly reactive to oxygen: Not indicated
Volatile: Not indicated

Analytical monitoring:

yes

Details on sampling:

Single samples for possible analysis were taken from all test concentrations and the control according to the schedule below. In addition, the filter containing the undissolved residue was kept for possible analysis.
Frequency: at t=0 h and t=48 h
Volume: 2.0 mL from the approximate centre of the test vessels
Storage: Samples were stored in a freezer until analysis.
At the end of the exposure period, the replicates were pooled at each concentration before sampling.
Additionally, single reserve samples of 2.0 mL were taken for possible analysis. If not used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Vehicle:

not specified

Details on test solutions:

The batch of Lowinox® 22IB46 tested was a white to cream coloured powder with a purity of 99.7% and not completely soluble in test medium at the concentrations tested. No correction was made for the purity/composition of the test item.
Preparation of test solutions started with a loading rate of 100 mg/L applying a three-day period of magnetic stirring to ensure maximum dissolution of the test item in test medium. Thereafter, the resulting mixture was filtered over a 0.45 μm membrane filter (Whatman; RC55). The obtained Saturated Solution (SS) was used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All final test solutions were clear and colourless.

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system
Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
Source: In-house laboratory culture with a known history.
Reason for selection: This system has been selected as an internationally accepted invertebrate species.
Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
Characteristics: For the test, selection of young daphnids with an age of < 24 hours, from parental daphnids older than two weeks.

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.
Medium: M7, as prescribed by Dr. Elendt-Schneider

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

No post exposure observation period specified in the study report.

Hardness:

The hardness: 180 mg/L expressed as CaCO3

Test temperature:

The temperature continuously measured in a temperature control vessel varied between 20 and 21°C during the test, and complied with the requirements as laid down in the study plan (18-22°C, constant within 2°C).

pH:

7.7 ± 0.3.

Dissolved oxygen:

oxygen: >3 mg/L at the end of the test.

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Lowinox® 22IB46: 4.6, 10, 22, 46 and 100% of a SS prepared at 100 mg/L.
Controls: Test medium without test item or other additives.

Details on test conditions:

Test duration: 48 hours
Test type: Static
Test vessels: 100 mL, all-glass
Medium: Adjusted ISO medium
Number of daphnids: 20 per concentration
Loading: 5 per vessel containing 80 mL of test solution
Light: 16 hours photoperiod daily
Feeding: No feeding
Aeration: No aeration of the test solutions.
Introduction of daphnids: Within 34 minutes after preparation of the test solutions.

Measurements and recordings
Immobility (including mortality): At 24 hours and at 48 hours.
Dissolved oxygen and pH: 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.

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.19 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Combined limit/range-finding test
No immobility was observed in the control or at the three lowest test concentrations during the test period. At the highest concentration, immobility increased to 10% and 85% after 24 and 48 hours, respectively. Therefore, the expected 48-hour EC50 was between 10 and 100% of the SS prepared at 100 mg/L.
Samples taken from 10 and 100% of the SS were analysed. The actual concentrations were 0.024 and 0.31 mg/L at the start of the test, respectively. The measured concentrations decreased to 54-79% of initial at the end of the test.
All test conditions were maintained within the limits prescribed by the study plan.

Final test
Measured concentrations
Analysis of the samples taken from the highest test concentration at the start of the final test showed a measured concentration of 0.19 mg/L. At the end of the exposure period, the measured concentration remained at 96% of the initial concentration. The initially measured concentration was thus used to describe the effect concentrations.

Immobility
No immobility was observed in the control or at any of the concentrations tested during the exposure period.
The responses recorded for the highest test group were not in agreement with the results of the combined limit/range-finding test. However, the concentration of test item dissolved in test medium in the undiluted SS during the combined limit/range-finding test was higher than during the subsequent final test. The achieved concentration in the final test was also more representative of the previously determined solubility limit in water. It can thus be concluded that the test item is not toxic to Daphnia at concentrations soluble in test medium.

Experimental conditions
These test conditions remained within the limits prescribed by the study plan (pH: 6.0-9.0, not varying by more than 1.5 units; oxygen: >3 mg/L at the end of the test).
The temperature continuously measured in a temperature control vessel varied between 20 and 21°C during the test, and complied with the requirements as laid down in the study plan (18-22°C, constant within 2°C).

Results with reference substance (positive control):

The actual responses in this reference test with K2Cr2O7 were within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was within the expected range of 0.30 to 1.0 mg/L. Hence, the sensitivity of the tested batch of Daphnia magna was comparable to the sensitivity of batches previously tested at Charles River Den Bosch.
The 24h-EC50 was 0.55 mg/L with a 95% confidence interval ranging from 0.50 to 0.62 mg/L.
The 48h-EC50 was 0.39 mg/L with a 95% confidence interval ranging from 0.33 to 0.44 mg/L. The historical ranges for the 48h-EC50 lie between 0.28 and 0.90 mg/L. The observed 48h-EC50 corresponds with this range.

Reported statistics and error estimates:

Not specified

Number of introduced daphnids and incidence of immobility in the combined limit/range-finding test

Time (h)	Replicate	Lowinox® 22IB46; % SS prep. at 100 mg/L
		Control	1.0	10	100
0	A B C D	5 5 5 5	5 5	5 5	5 5 5 5
	Total introduced	20	10	10	20
24	A B C D	0 0 (1) 0 0	0 0	0 0	0 1 1 0
	Total immobilized Effect %	0 0	0 0	0 0	2 10
48	A B C D	0 0 0 0	0 0	0 0	4 5 5 3
	Total immobilized Effect %	0 0	0 0	0 0	17 85

( ) between brackets: number of daphnia observed trapped at the surface of the test solutions. These organisms were re-immersed into the respective solutions before recording of mobility.

 

Number of introduced daphnids and incidence of immobility in the final test

Time (h)	Replicate	Lowinox® 22IB46; % SS prep. at 100 mg/L
		Control	4.6	10	22	46	100
0	A B C D	5 5 5 5	5 5 5 5	5 5 5 5	5 5 5 5	5 5 5 5	5 5 4* 5
	Total introduced	20	20	20	20	20	20
24	A B C D	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0
	Total immobilized Effect %	0 0	0 0	0 0	0 0	0 0	0 0
48	A B C D	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0	0 0 0 0
	Total immobilized Effect %	0 0	0 0	0 0	0 0	0 0	0 0

* During the final test, 4 instead of 5 daphnids were introduced in vessel C of the 100% SS test group.

 

Effect parameters

Parameter	Lowinox® 22IB46; Measured conc. (mg/L)
24h-EC50 48h-EC50	>0.19^*1 >0.19^*1

^*95% confidence intervals could not be determined

¹Analytically confirmed concentration in a saturated solution prepared at a loading rate of 100 mg/L.

 

pH and oxygen concentrations (mg/L) during the final test

Lowinox® 22IB46 % SS prep. at 100 mg/L	Start (t = 0 h)		End (t = 48 h)
	pH	O2	pH	O2
Control 4.6 10 22 46 100	7.8 7.7 7.7 7.7 7.7 7.9	9.1 9.1 9.1 9.2 9.1 9.1	8.2 8.2 8.2 8.1 8.1 8.1	8.8 8.7 8.7 8.8 8.9 8.9

 

Acute immobilization of daphnia after 24 and 48 hours in the reference test with potassium dichromate:

Potassium Dichromate Nominal conc. (mg/L)	Number Exposed	% immobile		Expected response (%) After 48 hours^1
		24 h	48 h	Minimal	Maximal
Control 0.10 0.18 0.32 0.56 1.0 1.8	20 20 20 20 20 20 20	0 0 0 0 55 100 100	0 0 0 30 95 100 100	0 0 0 0 0 40 100	10^2 10 10 30 100 100 100

¹Based on historical data of the previous years (n>60)

²A maximum response of 10% does not invalidate the results of the test.

Validity criteria fulfilled:

yes

Conclusions:

The 48h-EC50 exceeded an analytically confirmed concentration of 0.19 mg/L, being the concentration obtained in a saturated solution prepared at a loading rate of 100 mg/L, which was comparable to the solubility limit of the test item in water.

Executive summary:

Acute Toxicity Study in Daphnia magna with Lowinox® 22IB46.

 

The study procedures described in this report were based on the OECD guideline No. 202, 2004. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 440/2008, Part C.2, 2008 and the OECD series on testing and assessment number 23, 2000.

 

The batch of Lowinox® 22IB46 tested was a white to cream coloured powder with a purity of 99.7% and not completely soluble in test medium at the concentrations tested.

 

Preparation of test solutions started with a loading rate of 100 mg/L applying a three-day period of magnetic stirring to ensure maximum dissolution of the test item in test medium.

Thereafter, the resulting mixture was filtered and the obtained Saturated Solution (SS) used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All final test solutions were clear and colourless.

 

A final test was performed based on the results of a preceding combined limit/range-finding test. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to 4.6, 10, 22, 46 and 100% of a SS prepared at 100 mg/L. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

 

Analysis of the samples taken from the highest test concentration at the start of the final test showed a measured concentration of 0.19 mg/L. At the end of the exposure period, the measured concentration remained at 96% of the initial concentration. The initially measured concentration was thus used to describe the effect concentrations.

 

The study met the acceptability criteria prescribed by the study plan and was considered valid.

 

The 48h-EC50 exceeded an analytically confirmed concentration of 0.19 mg/L, being the concentration obtained in a saturated solution prepared at a loading rate of 100 mg/L, which was comparable to the solubility limit of the test item in water.

Description of key information

Key value determined in a GLP accredited laboratory study using Daphnia magna in accordance with OECD Guideline 202 and EU Method C2.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.19 mg/L

Additional information

Acute Toxicity Study in Daphnia magna with Lowinox® 22IB46.

 

The batch of Lowinox® 22IB46 tested was a white to cream coloured powder with a purity of 99.7% and not completely soluble in test medium at the concentrations tested.

 

Preparation of test solutions started with a loading rate of 100 mg/L applying a three-day period of magnetic stirring to ensure maximum dissolution of the test item in test medium.

Thereafter, the resulting mixture was filtered and the obtained Saturated Solution (SS) used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All final test solutions were clear and colourless.

 

A final test was performed based on the results of a preceding combined limit/range-finding test. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to 4.6, 10, 22, 46 and 100% of a SS prepared at 100 mg/L. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

 

Analysis of the samples taken from the highest test concentration at the start of the final test showed a measured concentration of 0.19 mg/L. At the end of the exposure period, the measured concentration remained at 96% of the initial concentration. The initially measured concentration was thus used to describe the effect concentrations.

 

The 48h-EC50 exceeded an analytically confirmed concentration of 0.19 mg/L, being the concentration obtained in a saturated solution prepared at a loading rate of 100 mg/L, which was comparable to the solubility limit of the test item in water.