sec-butylamine

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:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

TEST MEDIUM
All testing was performed using a synthetic medium. Chemical reagents used for the preparation of this dilution water were of “analytical grade”; it is prepared according to the protocol as described below to get the concentration specified in the test guideline:

The following solutions were prepared for the definitive test:
- CaCl2, 2H2O: 426.3 g per 1L of deionised water,
- MgSO4, 7H2O: 178.7 g per 1L of deionised water,
- NaHCO3: 93.9 g per 2 L of deionised water,
- KCl: 8.3 g of per 0.5L of deionised water,

All these solutions were then mixed with around 1450L of deionised water to get the following final recommended concentrations:
- CaCl2, 2H2O: 294 mg/L
- MgSO4, 7H2O: 123 mg/L
- NaHCO3: 64.7 mg/L
- KCl: 5.7 mg/L

TEST SOLUTIONS
RANGE-FINDING TEST
Tests solutions were prepared from dilutions of a stock solution: a known volume of test item (693 µL) was poured into a volumetric flask, the volume was then made up to 5 L with test medium. The solution was kept under high speed stirring at room temperature during approximately 15 minutes with a magnetic stir bar. No undissolved particles were observed, no filtration was thus necessary. In accordance with the Sponsor's representative, the pH of the test item stock solutions was measured at pH 10.4 and adjusted to pH 7.4.

Test solutions were renewed every day throughout the 96h test period (semi-static test) from a new test item stock solution prepared daily as described above.

Fish were exposed to the following nominal test concentrations: 1.0, 10.0, 50.0 and 100.0 mg/L. One tank was prepared at each test concentration; each tank contained 7 fish.

DEFINITIVE TEST
Based on the results of the range-finding test, the definitive test was conducted under semi-static conditions at the following nominal test concentrations: 50.0, 100, 200, 400 and 800.0 mg/L. The range of concentrations was obtained from dilutions of a stock solution prepared daily as follows: a known volume of test item (6,925 mL) was poured into a volumetric flask, the volume was then made up to 5 L with test medium. The solution was kept under high speed stirring at room temperature during a few minutes with a magnetic stir bar. No undissolved particles were observed, no filtration was thus necessary. The pH of the test item stock solution was adjusted to pH 7.7 before dilutions.

One tank was prepared at each test concentration and the control group; each tank contained 7 fish.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

The test organism was Danio rerio (Teleostei, Cyprinidae).

- Preliminary range-finding test: the batch 17/Br/01/1 was obtained on the 03 February 2017 from La Grande Rivière France (69490 Saint-Forgeux, France)
- Definitive test: the batch 17/Br/03/1 was obtained on the 15 November 2017 from AMAGEN UMS (91190 Gif sur Yvette, France)

All fish were in good health and free from any apparent malformation. Mortality of the batch was less than 5% in the week preceding the start of the study. No disease treatments were administered throughout holding and testing.

Holding was maintained within the laboratory at a temperature of 21-25°C in glass tanks. A light cycle of 12 h light and 12 h dark was applied, illumination being provided by fluorescent tubes (intensity between 400 and 800 lux at the surface of the tanks). Tanks were aerated to ensure that the dissolved oxygen concentration is at least 60% of air saturation value in holding tanks and in test tanks. During holding, fish were fed twice per day with ground flake food TetraMin®.

The fish were not fed for a period of 24h prior to test commencement or throughout the duration of the test

Fish were added to the test tanks within 30 min of the completion of preparation of the test solutions. All test fish were weighed prior to the test and a representative number of test fish batch (10 at random) was measured after the test to assess compliance with guideline criteria.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

250 mg/L as CaCO3.

Test temperature:

min.: 22.7°C, max.: 23.1°C.

pH:

7.8 - 8.1

Dissolved oxygen:

91.3 - 103.6% of saturation.

Nominal and measured concentrations:

Nominal: control ; 50 ; 100 ; 200 ; 400 ; 800 mg/L
Measured concentrations (geometric means): control ; 58.9 ; 113.4 ; 210.5 ; 450.6 ; 880.1 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 5-L glass aquarium with lid.
- Fill volume: 2.5 L
- Renewal rate of test solution: once a day (semi-static).
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1

OTHER TEST CONDITIONS
- Adjustment of pH: yes, in stock solution
- Photoperiod: 15L:9D
- Light intensity: 400-800 lx at water surface

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: 0 ; 1 ; 10 ; 100 mg/L
- Results used to determine the conditions for the definitive study: LC0 > 100 mg/L

Reference substance (positive control):

yes

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 880.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Remarks on result:

other: see remarks

Remarks:

2 dead fish out of 7 at 800 mg/L

Details on results:

RANGE-FINDING TEST
The appearance of the test solutions was visually checked at the beginning and at the end of the test. Solutions were found to be clear, no precipitation was observed at the end of the test. Dissolved oxygen and pH measurements, carried out at each observation time did not fall below or above the recommended limits. The temperature was maintained in a range from 21°C to 25°C and remaining constant within ± 2°C during the test: mean measured temperature: 23.0°C, min.: 22.7C, max.: 23.5°C. The numbers of dead fish recorded over the test period are presented in Table . No significant visible abnormalities were reported.
The mean length of fish, measured on 10 randomly organisms, was 3.7 cm (min. 3.4, max. 3.9). The measured concentrations in tanks showed that the substance was unstable at all concentrations in the test medium during 96 hours. It was thus decided to perform the definitive test under semi-static conditions with daily renewal.


DEFINITIVE TEST
The appearance of the test solutions was visually checked at the beginning and at the end of the test. Solutions were found to be clear, no precipitation was observed at the end of the test. Dissolved oxygen and pH measurements, carried out at each observation time, were within the range prescribed by OECD 203 TG for zebrafish. The total hardness was determined at 250 mg CaCO3/L. The temperature was maintained in a range from 21°C to 25°C and remaining constant within ± 2°C during the test: mean measured temperature: 22.9°C, min.: 22.7°C, max.: 23.1°C.
The numbers of dead fish recorded over the test period are presented in Table 1. Measured test item concentrations in the definitive test are presented in Table 2. Chemical analysis of test samples taken indicated that measured concentrations of the test item did not remained within 80-120% of the nominal concentrations and were not maintained over the 96h test period (i.e. deviation >20%). Nevertheless, test item concentration always remained within 20% of the initially measured concentration. Based on these results, the exposure concentrations were based on the arithmetic mean of the concentrations measured in fresh solutions. The mean length of fish, measured on 10 randomly organisms, was 2.5 cm (min. 1.8, max. 3.0).

Results with reference substance (positive control):

The sensitivity of the test system and the methodology are evaluated with every batch of fish by performing an acute toxicity test on potassium dichromate. The values of 24h-LC50 obtained with the batches used were as follows:

- Preliminary range-finding test: Batch 17/Br/01/1: 24h-LC50 = 205.8 mg/L
- Definitive test: Batch 17/Br/03/1: 24h-LC50 = 192.0 mg/L

These results are consistent with the laboratory control quality chart.

Reported statistics and error estimates:

Since only two fish out of seven died at the highest concentration tested, statiscal analysis was considered unnecessary.

Sublethal observations / clinical signs:

Table 1: Cumulative lethality (Definitive test)

Test solution (mg/L)	Cumulative dead fish					Mortality at the end of the period
	T0	T24h	T48h	T72h	T96h
Control	0	0	0	0	0	0,0%
50,0	0	0	0	0	0	0,0%
100.0	0	0	0	0	0	0,0%
200.0	0	0	0	0	0	0,0%
400.0	0	0	0	0	0	0,0%
800.0	0	0	0	0	2	28,6%

 

Table 2: Test item determination – Concentrations used for statistical analysis (Definitive test)

Nominal concentration (mg/L)	Test item measured concentrations(mg/L)												Conc. used for statistical analysis. (mg/L)*
	T0/T24			T24/T48			T48/72			T72/96
	fresh	old	eviation T0/T24	fresh	old	Deviation T24/T48	fresh	old	Deviation T48/T72	fresh	old	Deviation T72/T96
Control	<DL	<DL	NA	<DL	<DL	NA	<DL	<DL	NA	<DL	<DL	NA	NA
50,0	57,0	60,1	5,4	68,8	63,7	-7,5	51,9	53,2	2,5	56,5	61,5	8,7	58,9
100.0	110,1	110,8	0,6	139,6	121,1	-13,2	102,5	101,6	-0,9	108,2	117,4	8,6	113,4
200.0	209,8	240,0	14,3	227,1	215,5	-5,1	197,8	197,1	-0,4	190,5	210,8	10,7	210,5
400.0	456,8	507,6	11,1	513,7	411,1	-20,0	430,9	392,4	-8,9	440,7	465,6	5,7	450,6
800.0	861,0	1049,5	21,9	971,6	891,0	-8,3	808,5	808,5	0,0	804,9	874,3	8,6	880,1

< DL 0.89 mg/L): concentration lower than the Detection Limit

< QL (2.98 mg/L): concentration lower than the Quantification Limit

NA: Not Applicable

*Concentrations extrapolated as arithmetical means of measured concentrations in fresh solutions.

Table 3: Measured pH (Definitive test)

Test solution (mg/L)	T0	T24		T48		T72		T96
	fresh sol.	old sol.	fresh sol.	old sol.	fresh sol.	old sol.	fresh sol.	old sol.
Control	8,0	7,8	7,9	7,8	7,9	7,9	8,1	7,8
50,0	8,0	7,9	7,9	7,9	8,0	7,9	8,0	7,9
100.0	7,9	7,8	8,0	7,9	8,0	7,9	8,0	7,9
200.0	7,9	7,8	7,9	7,8	8,0	7,9	8,0	7,8
400.0	7,9	7,8	7,9	7,8	7,9	7,8	7,9	7,8
800.0	7,8	7,8	7,8	7,8	7,8	7,8	7,8	7,8

Table 4: Measured dissolved oxygen expressed in % of air saturation (Definitive test)

Test solution (mg/L)	T0	T24		T48		T72		T96
	fresh sol.	old sol.	fresh sol.	old sol.	fresh sol.	old sol.	fresh sol.	old sol.
Control	97,7	93,1	101,0	100,1	103,2	91,3	101,0	93,6
50,0	99,6	94,8	101,4	100,3	103,6	95,7	101,1	94,1
100.0	99,3	94,3	100,4	100,4	103,0	95,2	100,9	94,2
200.0	99,3	93,7	100,5	98,5	102,9	96,1	100,8	92,6
400.0	98,9	92,8	99,9	98,7	102,8	93,8	100,3	91,4
800.0	98,8	95,7	99,7	100,7	102,9	94,7	100,2	92,5

Validity criteria fulfilled:

yes

Conclusions:

The 96h-LC50 of fish is higher than 880.1 mg/L for 2-butanamine.

Executive summary:

This study was designed to determine the effects of the test item to Danio rerio over a 96h period with static conditions, according to the OECD 203 Guideline. The tests solutions were prepared from dilutions of a stock solution prepared freshly every day. pH was adjusted where relevant. 

Chemical analysis of test samples taken indicated that measured concentrations of the test item did not remain within 80-120% of the nominal concentrations and were not maintained over the 96h test period (i.e. deviation >20%). Consequently, geometric means of measured concentrations were used for the statistical analysis. From the results obtained it can be concluded the 96h-LC50 is higher than the highest tested concentration, i.e. 880.1 mg/L (expressed as geometric mean of concentrations measured in all samples). .

The definitive test met the validity criteria of the test guideline detailed as follows:

 § The mortality in the control did not exceed 10% at the end of the test;

§ The dissolved oxygen in the test tanks remained above 60% of the air saturation value throughout the test;

§ The pH did not vary by more than 1 unit.

Description of key information

The toxicity of neutralized solutions of 2 -butanamine was tested on Danio rerio. It was observed that 96h-LC50 was higher than 860 mg/L.