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

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

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 203 (Fish, Acute Toxicity Test)

Version / remarks:

OECD guidelines for Testing of Chemicals, guideline No. 203: "Fish Acute Toxicity Test", Adopted 17 July, 1992.

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:

During the final test 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, t=24 h and t=96 h
Volume: 2.0 mL from the approximate centre of the test vessels
Storage: Samples were stored in a freezer until analysis.
Additionally, single reserve samples of 2.0 mL were taken from all test solutions for possible analysis. If not already 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):

Cyprinus carpio

Details on test organisms:

Test System
Species: Carp (Cyprinus carpio, Teleostei, Cyprinidae) Linnaeus, 1758
Source: Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, The Netherlands.
Mean length: Range-finding test: 2.9 ± 0.3 cm
Final test: 2.5 ± 0.2 cm
Mean weight: Range-finding test: 0.77 ± 0.21 g
Final test: 0.20 ± 0.07 g
Characteristics: F1 from a single parent-pair bred in UV-treated water.
Reason for selection: This system has been selected as an internationally accepted species.
Total fish used: 51

Holding
Quarantine/Acclimatisation: At least 12 days after delivery
Medium: Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands)
Measurements: Conductivity, pH, nitrate, nitrite and ammonia concentration: once a week. Temperature: continuous. In addition, pH and temperature were measured before transferring the fish to the test system.
Water quality parameters: Were kept within the optimum limits for the respective fish species.
Feeding: Daily with pelleted fish food (Essence (300-500 um), Coppens International bv, Helmond, The Netherlands)
Validity of batch: In the batch of fish used for the test, mortality during the seven days prior to the start of the test was less than 5%.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

Post exposure observation period not specified in the study report.

Hardness:

180 mg CaCO3 per litre

Test temperature:

20-24°C, constant within 2°C;

pH:

7.7 ± 0.3.

Dissolved oxygen:

oxygen > 60% of air saturation

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

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

Details on test conditions:

Test duration: 96 hours
Test type: Static
Test vessels: 3.5 litres, all-glass, containing 3 litres of test solution
Test medium: Adjusted ISO medium with a hardness of 180 mg CaCO3 per litre and a pH of 7.7 ± 0.3.
Number of fish: 7 per concentration
Loading: 0.47 g fish/Litre, i.e. 7 fish per 3 litres of test medium
Illumination: 16 hours photoperiod daily
Aeration: The test media were not aerated during the test.
Feeding: No feeding from 24 hours prior to the test and during the total test period
Introduction of fish: Within 35 minutes after preparation of the test media from a holding tank with comparable water quality parameters and pH and temperature differences between test and holding tank media of less than 0.5 unit and 0.5°C.
Euthanasia: At the end of the test the surviving fish were rapidly killed by exposing them to ca. 1.2% ethylene glycol monophenylether in water.

Measurements and recordings
Mortality and other effects: At 2, 24, 48, 72 and 96 hours following the start of exposure. In addition, every afternoon from day 0 (except for day 1) and every morning from day 1 to observe for any dead or severely distressed fish. Dead fish were removed when observed.
Dissolved oxygen content, pH and temperature: Daily in all vessels with surviving fish, beginning at the start of the test (day 0).

Reference substance (positive control):

yes

Remarks:

pentachlorophenol (PCP)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.064 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Range-finding test
No mortality or severe clinical effects were observed in the dilutions representing 1.0 and 10% of the SS during the test period (only effects; hyperactive fish after 3 h in 10%SS). Fish exposed to the undiluted (100%) SS all died within the first three days of exposure after showing loss of righting reflexes. Based on these results samples taken from the solutions containing 10 and 100% SS were analysed. The concentrations measured at the start of the test were 0.024 and 0.31 mg/L for 10 and 100% SS, respectively. These concentrations decreased by approximately 40% during the first 24-hour test period and by 54-65% over the total test period of 96 hours.
Considering the low water solubility (0.203 mg/L; CRL project 512734), the absence of any additional new peaks in the chromatograms and the non-linear decrease in concentration during the test period it was expected that the decrease was due to the limited solubility instead of test item instability in test medium. It was therefore decided not to renew test solutions in the final test.
The expected LC50 was between test groups containing 10 and 100% of a SS prepared at 100 mg/L. All test conditions were maintained within the limits prescribed by the study plan.

Final test
Measured concentrations
Analysis of the samples taken at the start of the final test showed measured concentrations of 0.017, 0.025, 0.045, 0.15 and 0.21 mg/L in the test groups containing 4.6, 10, 22, 46 and 100% SS, respectively. Hence, the measured concentration in the undiluted SS was in agreement with the expected water solubility. After 24 hours of exposure the test item concentrations measured decreased by 21-55% below initial without any relation to the concentration. After 96 hours concentrations had decreased slightly more. Based on these results, the average exposure concentrations were calculated (see Table 2). The range tested based on average measured concentrations was 0.014, 0.014, 0.029, 0.060 and 0.18 mg/L.

Mortality and other effects
The responses recorded in this test allowed for reliable determination of an LC50 and were in agreement with what was expected based on the results of the range-finding test.

Experimental conditions
All test conditions remained within the ranges prescribed by the study plan (pH: 6.0-8.5, constant within 1 unit; temperature 20-24°C, constant within 2°C; oxygen > 60% of air saturation).

Results with reference substance (positive control):

Under the conditions of the present test all carp exposed to a PCP concentration of 0.46 mg/l died within 24 hours. One fish died at 0.22 mg/l and no mortality occurred at 0.10 mg/l. The 96h-LC50 for carp exposed to PCP was 0.24 mg/l. This effect was already reached within 48 hours of exposure.
The range of the 96h-LC50 for carp is generally between 0.10 and 0.46 mg/l based on historical data of reference tests performed approximately every 3 months from April 1988 until the end of 2000, and annually since then. Hence, the sensitivity of carp originating from the present batch for PCP falls within the range of sensitivities generally observed during the past years.

Reported statistics and error estimates:

Not specified

Sublethal observations / clinical signs:

Incidence of mortality and total mortality during the range-finding test

Lowinox® 22BI46 % SS prep. at 100 mg/L	Initial number of fish	Cumulative mortality					Total Mortality (%)
		3h	24h	48h	72h	96h
1.0 10 100	3 3 3	0 0 0	0 0 1	0 0 2	0 0 3	0 0 3	0 0 100

 

Measured concentrations versus nominal concentrations

Lowinox® 22IB46 % SS prep. at 100 mg/L	Measured concentration (mg/L)			Average (mg/L)
	t=0h	t=24h	t=96h
4.6 10 22 46 100	0.0171 0.0252 0.0452 0.149 0.208	0.013 0.015 0.0329 0.0666 0.164	0.013 0.010 0.0194 0.0309 nm	0.014 0.014 0.029 0.060 0.18

nm not measured (all fish had died after 24h)

 

Incidence of mortality and total mortality during the final test

Lowinox® 22BI46 % SS prep. at 100 mg/L	Initial number of fish	Cumulative mortality					Total Mortality (%)
		3h	24h	48h	72h	96h
Control 0.014 0.014 0.029 0.060 0.18	7 7 7 7 7 7	0 0 0 0 0 0	0 0 0 0 0 7	0 0 0 0 2 7	0 0 0 0 3 7	0 0 0 0 3 7	0 0 0 0 43 100

 

Clinical effect observations during the final test

Lowinox® 22IB46 Average conc. (mg/L)	Time of recording (hours)	Specification of effects	Relative number
Control, 0.014, 0.014, 0.029         0.060     0.18	0-96 24   48 72-96   24 48 72-96 2	No effects Loss of righting reflex, swimming at bottom tank Swimming at bottom of tank Loss of righting reflex, swimming at bottom tank Loss of righting reflex, swimming at bottom tank Swimming vertical at bottom tank, discoloured Loss of righting reflex, swimming at bottom tank Loss of righting reflex, swimming at bottom tank Loss of righting reflex, swimming at bottom tank Loss of righting reflex, hyperactive	7/7 3/7 4/7 7/7 6/7 1/7 7/7 5/5 4/4 7/7

 

Effect parameters

Parameter	Lowinox® 22IB46 Average conc. (mg/L)	95% confidence interval (mg/L)
24h-LC50 48h-LC50 72h-LC50 96h-LC50	0.10 0.073 0.064 0.064	0.060-0.18 0.051-nd 0.047-0.16 0.047-0.16

nd: not determined

 

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

Lowinox® 22IB46 Average conc. (mg/L)	Day 0		Day 1		Day 2		Day 3		Day 4
	pH^1	O2	pH	O2	pH	O2	pH	O2	pH	O2
Control 0.014 0.014 0.029 0.060 0.18	7.7 7.7 7.7 7.7 7.6 7.7	9.1 9.1 9.1 9.2 9.1 9.2	7.7 7.6 7.6 7.6 7.6 7.6	7.0 6.8 7.0 7.0 6.8 7.9	8.2 8.1 8.0 8.0 7.9 -	7.3 6.7 6.6 6.7 6.5 -	7.8 7.6 7.6 7.6 7.5 -	6.9 6.3 6.5 6.3 6.3 -	8.0 7.9 7.7 7.6 7.5 -	6.7 6.2 6.4 6.4 6.2 -

¹pH of culture medium was: 7.8

 

Temperatures (°C) measured during the final test

Lowinox® 22IB46 Average conc. (mg/L)	Day 0^1	Day 1	Day 2	Day 3	Day 4
Control 0.014 0.014 0.029 0.060 0.18	21.5 21.5 21.5 21.5 21.5 21.6	20.9 21.0 20.9 20.9 20.9 20.9	20.9 20.8 20.8 20.8 20.8 -	20.9 20.9 20.8 20.9 20.8 -	21.1 21.2 21.2 21.2 21.2 -

¹Temperature of culture medium was: 21.7°C and test solutions, except the highest test concentration, were heated before exposure of fish to 21.5°C.

 

Incidence of mortality observed in the reference study:

Concentration PCP (mg/l) Nominal	Initial Number Of Fish	Cumulative number of dead fish recorded at various time points after start of exposure					Total Mortality (%)
		4h	24h	48h	72h	96h
0.10	5	0	0	0	0	0	0
0.22	5	0	0	1	1	1	20
0.46	5	0	5	5	5	5	100

 

Validity criteria fulfilled:

yes

Conclusions:

The 96h-LC50 was 0.064 mg/L based on average measured concentrations (95% confidence interval between 0.047 and 0.16 mg/L) and was already reached after 72 hours of exposure.

Executive summary:

96-Hour Acute Toxicity Study in Carp with Lowinox® 22IB46.

 

The study procedure described in this report was based on the OECD guideline No. 203, 1992. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 440/2008, Part C.1, 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 range-finding test. Seven carp per test group were exposed to an untreated control and to Lowinox® 22IB46 test groups containing 4.6, 10, 22, 46 and 100% SS prepared at a loading rate of 100 mg/L. Samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 96 hours of exposure.

 

Analysis of the samples taken at the start of the final test showed measured concentrations of 0.017, 0.025, 0.045, 0.15 and 0.21 mg/L in the test groups containing 4.6, 10, 22, 46 and 100% SS, respectively. Hence, the measured concentration in the undiluted SS was in agreement with the expected water solubility. After 24 hours of exposure the test item concentrations measured decreased by 21-55% below initial without any relation to the concentration. After 96 hours concentrations had decreased slightly more. Based on these results, the average exposure concentrations were calculated and corresponded with 0.014, 0.014, 0.029, 0.060 and 0.18 mg/L.

 

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

 

The 96h-LC50 was 0.064 mg/L based on average measured concentrations (95% confidence interval between 0.047 and 0.16 mg/L) and was already reached after 72 hours of exposure.

Description of key information

Key value determined in a GLP accredited laboratry study using Carp in accordance with OECD Guideline 203 and EU Method C1.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.064 mg/L

Additional information

96-Hour Acute Toxicity Study in Carp 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 range-finding test. Seven carp per test group were exposed to an untreated control and to Lowinox® 22IB46 test groups containing 4.6, 10, 22, 46 and 100% SS prepared at a loading rate of 100 mg/L. Samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 96 hours of exposure.

 

Analysis of the samples taken at the start of the final test showed measured concentrations of 0.017, 0.025, 0.045, 0.15 and 0.21 mg/L in the test groups containing 4.6, 10, 22, 46 and 100% SS, respectively. Hence, the measured concentration in the undiluted SS was in agreement with the expected water solubility. After 24 hours of exposure the test item concentrations measured decreased by 21-55% below initial without any relation to the concentration. After 96 hours concentrations had decreased slightly more. Based on these results, the average exposure concentrations were calculated and corresponded with 0.014, 0.014, 0.029, 0.060 and 0.18 mg/L.

 

The 96h-LC50 was 0.064 mg/L based on average measured concentrations (95% confidence interval between 0.047 and 0.16 mg/L) and was already reached after 72 hours of exposure.