4,4'-methylenebis[N-sec-butylaniline]

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:

08 Jun 2018 - 18 Jul 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1992

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: all test concentrations and Control
- Sampling method: 2.0 mL from the approximate centre of the vessels,at the start of the test and after 72 h from the freshly prepared solutions, at the first renewal (t=24 h) and the end of the test from the 24-h old solutions.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Saturated Solution. 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. The obtained mixture was allowed to settle for a period of three hours. Thereafter, the aqueous Saturated Solution (SS) was collected by means of siphoning and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. 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):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: Zodiac, proefacc. "De Haar Vissen", Wageningen University and Research Centre, The Netherlands
- Length at study initiation: 2.9 +/- 0.1 cm (Final test)
- Weight at study initiation: 0.30 +/- 0.05 g (Final test)
- Method of breeding: F1 from a single parent-pair bred in UV-treated water
- Total fish used: 51

HOLDING
- Quarantine/Acclimatisation: At least 12 days after delivery
- Medium: Adjusted ISO medium, formulated using tap-water purified by reverse osmosis
- Feeding: Daily with pelleted fish food (Cyprico Crumble Excellent (300-500 um))
- 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:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

180 mg CaCO3/L

Test temperature:

21 - 22 °C

pH:

7.8 - 8.1

Dissolved oxygen:

Day 0: fresh 8.3-8.7 mg/L
Day 1: old 7.0-7.6 mg/L; fresh 8.4-8.8 mg/L
Day 2: old: 7.6-8.3 mg/L; fresh 8.6-8.9 mg/L
Day 3: old: 7.5-8.4 mg/L; fresh 9.2-9.9 mg/L
Day 4: old 7.1-8.0 mg/L

Nominal and measured concentrations:

Nominal: 10, 18, 32, 56, 100% of saturated solution prepared at loading rate 100 mg/L
Measured concentrations: Concentrations measured in the freshly prepared solutions ranged between 0.080 and 0.83 mg/L during the first renewal, and between 0.075 and 0.73 mg/L, during the last renewal period. Measured concentrations decreased to 45-73% of initially measured at the end of the first renewal and to 43-70% at the end of the last renewal period. Therefore, average exposure concentrations were calculated. See Table 1 in 'Any other information on results' for details.

Details on test conditions:

TEST SYSTEM
- Test vessel: 6.5 litres, all-glass, containing 4 litres of test solution
- Test type: semi-static, with daily renewal of test solutions
- Aeration: no aeration
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.53 g fish/L, i.e. 7 fish per 4 litres of test medium

TEST MEDIUM / WATER PARAMETERS
- Test medium: Adjusted ISO medium
- Culture medium different from test medium: no
- Intervals of water quality measurement: Dissolved oxygen content, pH and temperature were measured daily in all vessels, beginning at the start of the test (day 0).

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h daily
- Feeding: No feeding from 24 hours prior to the test and during the total test period
- Introduction of fish: Within 17 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 1.0 unit and 1.0°C, respectively.

TERMINAL PROCEDURES
- 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.

EFFECT PARAMETERS MEASURED
- Mortality and other effects: At 2.5, 24, 48, 72 and 96 hours following the start of exposure. In addition, every afternoon from day 0 to observe for any dead or severely distressed fish.

TEST CONCENTRATIONS
- Range finding study test concentrations: 1.0, 10 and 100% of saturated solution prepared at a loading rate of 100 mg/L.
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Pentachlorophenol (performed April 2018)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 0.61 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality (fish)

Remarks on result:

other: No mortality observed; 0.61 mg/L is considered the maximum solubility of test item in test medium at a loading rate of 100 mg/L

Details on results:

- Small responses at the retention time of the test item were detected in the chromatograms of the blank QC samples analyzed on 20 Jun 2018. It was considered to derive from carry-over in the analytical system since similar responses were found in the analytical blanks. The maximum contribution to the QC samples at the lowest concentration level was 1.3% based on area. It was considered that it has no significant effect on the results of the test samples.
- The mean accuracies of the QC samples containing test item fell within the criterion of 70-110%, except for QC level of 0.001 mg/L analyzed on 17 Jul 2018 (where the mean accuracy was 140%). This was considered to have no impact on the results of the study as a target concentration of the samples fell within the concentration range of higher QC samples.
- No mortality was observed at any of the concentrations tested during the test period. At the two highest test concentration, all fish showed increasingly changed behaviour with progressing exposure time. See table 2 in 'Any other information on results' for details on clinical effects observed during the test.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Mortality: 0% at 0.10 mg/L PCP; 80% at 0.22 mg/L PCP; 100% at 0.46 mg/L PCP
- LC50: 0.17 mg/L, 95% confidence interval 0.13-0.23 mg/L
- The range of the 96h-LC50 for zebra-fish is generally between 0.10 mg/L and 0.46 mg/L based on historical data of reference tests performed by the Test Facility. Hence, the sensitivity of zebra-fish originating from the present batch for PCP falls within the range of sensitivities generally observed during the past years.

Reported statistics and error estimates:

No LC50 could be calculated because the test item proved to be non-toxic (LC50 > maximum soluble concentration).

Sublethal observations / clinical signs:

Table 1 Measured Concentrations and the Calculated Average Exposure Concentration 

4,4’-methylenebis-N-sec-butylaniline %SS prep. at 100 mg/L	Measured concentrations (mg/L)				Average exposure conc. (mg/L)
	t=0h fresh	t=24h old	t=72h fresh	t=96h old
10	0.080	0.037	0.075	0.039	0.054
18	0.16	0.07	0.16	0.067	0.10
32	0.28	0.17	0.23	0.17	0.21
56	0.52	0.38	0.40	0.28	0.39
100	0.83	0.50	0.73	0.45	0.61

Table2: Clinical Effects Observed During the Final Test

4,4’-methylenebis-N-sec-butylaniline Average conc. (mg/L)	Time of recording (hours)	Specification of effects	Relative number
0.39	96	Loss of equilibrium Swimming at the bottom of the test vessel	1/7 2/7
0.61	48	Swimming at the bottom of the test vessel	3/7
	72	Swimming at the bottom of the test vessel	7/7
	96	Loss of equilibrium Swimming at the bottom of the test vessel	1/7 6/7

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks' for details on validity criteria.

Conclusions:

The 96h-LC50 was beyond the range tested, i.e. exceeded an average exposure concentration of 0.61 mg/L being considered to represent the maximum solubility of the test item in test medium at a loading rate of 100 mg/L.

Executive summary:

In a study performed in accordance with OECD 203 (1992) and according to GLP principles, the acute toxicity of the substance to zebrafish (Danio rerio) was investigated. In semi-static test, zebrafish were exposed for 96 hours to an untreated control and to 10, 18, 32, 56 and 100% of a Saturated Solution prepared at a loading rate of 100 mg/L (7 fish per treatment group).

Measured concentrations decreased to 45 -73 % of initial at the first renewal and to 43 -70% of initial at the end of the last renewal period. Average exposure concentrations were calculated to be 0.054, 0.10, 0.21, 0.39 and 0.61 mg/L.

No mortality was observed in any of the test groups. Behavioural changes were observed in all fish in the two highest test concentrations at 72 and 96 h.

The 96h-LC50 of the substance to Danio rerio is concluded to be beyond the range tested, i.e. exceeded an average exposure concentration of 0.61 mg/L, being considered to represent the maximum solubility of the test item in test medium at a loading rate of 100 mg/L.

The test met all validity criteria and is considered to be reliable without restriction.

Description of key information

The 96h-LC50 was beyond the range tested, i.e. exceeded an average exposure concentration of 0.61 mg/L being considered to represent the maximum solubility of the test item in test medium at a loading rate of 100 mg/L.

Additional information

In a study performed in accordance with OECD 203 (1992) and according to GLP principles, the acute toxicity of the substance to zebrafish (Danio rerio) was investigated. In a semi-static test, zebrafish were exposed for 96 hours to an untreated control and to 10, 18, 32, 56 and 100% of a Saturated Solution prepared at a loading rate of 100 mg/L (7 fish per treatment group).

Measured concentrations decreased to 45 -73 % of initial at the first renewal and to 43 -70% of initial at the end of the last renewal period. Average exposure concentrations were calculated to be 0.054, 0.10, 0.21, 0.39 and 0.61 mg/L.

No mortality was observed in any of the test groups. Behavioral changes were observed in all fish in the two highest test concentrations at 72 and 96 h.

The 96h-LC50 of the substance to Danio rerio is concluded to be beyond the range tested, i.e. exceeded an average exposure concentration of 0.61 mg/L, being considered to represent the maximum solubility of the test item in test medium at a loading rate of 100 mg/L.