sec-butylamine

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

Osmotically filtered water was used to prepare the dilution water. As the use of a sealed exposure system in the algal growth inhibition test will result in culture growth being limited by CO2 depletion and increasing pH, the test medium was completed with a buffer solution ("Hepes medium" at 1 mM with NaHCO3 at 0.6 g/L) and sodium bicarbonate. Chemical reagents used for the preparation of dilution water were of “analytical grade”.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

The test organism used for the study was Pseudokirchneriella subcapitata, Strain No. CCAP 278/4, supplied by the Culture Centre of Algae and Protozoa (Ambleside, UK). Transfers of P. subcapitata were made regularly to provide suitable subcultures. The algae were cultivated under standardized conditions as described in Annex 2 of the OECD 201 guideline. The quality of the stock culture was checked for the absence of micro-organisms and deformed or abnormal cells under microscopic observation before use. Three days before the start of the exposure, two pre-cultures were prepared by inoculating sufficient cells from the algal stock culture to the growth medium to obtain a low cell density, e.g 10000 cells/mL for pre-culturing, in order to maintain exponential growth until the start of the test. The pre-cultures were incubated under the same conditions as those used for the test cultures. Only one of the two pre-cultures was used to inoculate the test flasks for the study. The second one was only used if the first one was damaged. At the beginning of the test, the cell density of the pre-culture was determined. The result was used to calculate the volume to be introduced into each test flask in order to obtain an initial cell concentration of 5000 or 10000 cells/mL as recommended in the OECD 201 guideline.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

Not measured but expected to have been maintained within 22-24°C (incubation in a thermically controlled chamber).

pH:

8.1 - 8.9 (min-max)

Dissolved oxygen:

8.4 - 10.5 mg/L (min-max)

Nominal and measured concentrations:

Nominal: control ; 0.16 ; 0.32 ; 0.64 ; 1.24 ; 2.48 ; 5 mg/L
Measured: did not deviate by more than 20% from nominal concentrations, neither at 0h and 72 h.

Details on test conditions:

RANGE-FINDING TEST
The substance was known to be volatile in the test conditions, appropriate procedures were thus chosen in accordance with the Study Monitor to design the test conditions: glass bottles of 120 mL nominal capacity filled up to 115 mL, tightly closed with bungs and without headspace were used.
Tests solutions were prepared from dilutions of a stock solution: a known volume of test item (138.5 µL, i.e. 100 mg according to the test item density mentioned in the safety datasheet joined to the test item sample received at the test facility) was poured into a volumetric flask, the volume was then made up to 1 L with test medium. The solution was kept under high speed stirring at room temperature during approximately 20 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 9.3 and adjusted to pH 7.7.
Algae were exposed to a series of the following series of nominal test concentrations: 1.0, 5.0, 10.0, 50.0 and 100.0 mg/L. An inoculated control flask was prepared and incubated under the same conditions, with no test item. Three vessels were prepared at each test concentration and six vessels for the control group; each vessel was inoculated with ca 10000 cells/mL P. subcapitata.

DEFINITIVE TEST
Based on the results of the range-finding test, the definitive test was conducted at the following series of nominal test concentrations: 0.16, 0.32, 0.64, 1.24, 2.48 and 5.00 mg/L. The range of concentrations was obtained from dilutions of the stock solution prepared as follows: a known volume of test item (34.64 µL, i.e. 25 mg according to the test item density) was poured into a volumetric flask, the volume was then made up to 250 mL with test medium. The solution was kept under high speed stirring at room temperature during around 10 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 7.5 before dilutions.
In addition, as chemical analysis of test samples conducted during the preliminary range-finding test indicated that measured concentrations of the test item remained within 80-120% of the nominal concentrations and were maintained over the 72h test period (i.e. deviation <20%), it was decided to keep a significant headspace in the test flask in order to ensure a good homogenization of the algae cells.
Three vessels were prepared at each test concentration and six vessels for the control group; each vessel was inoculated with ca 104 cells/mL P. subcapitata.

OBSERVATIONS
Algal cell concentrations were measured in each flask at 24, 48 and 72 h using flow cytometry (Guava easyCyte™ flow cytometer Merck Millipore). Cell concentrations were determined using 96 wells single use microplates and a laser beam at 488 nm. After 24, 48h and 72h of incubation, aliquot of 200 µL was sampled from each inoculated test flask and pipetted into a microplate. Time between sampling and measurement is approximately 15 – 30 min. The cytometer is calibrated every week using the Guava check kit. Before each measurement, settings were adjusted. Non-algal particles were excluded from the analysis by setting an acquisition value threshold. This threshold was set up after analysis of cytograms with no threshold where a clear discrimination was observed between the algal population and the other events. Therefore, only the events with the same size than alga were used to assess the toxic effects. The number of events counted was set up to 1500 to 3000 depending on the algal population. To minimize the number of coincident events, the analysed cell concentration was less than 500 cells/µL. Data processing was carried out using “Insight Software” (Guava easyCyte™ flow cytometer Merck Millipore).

Reference substance (positive control):

yes

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.87 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

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. Microscopic observation confirmed that the algae did not appeared "normal" at the end of the test: the normal shape of P. subcapitata algae is a crescent shaped cell with an average length of 5-10 µm: in this test algae cells were found to be inflated and distorted.
Chemical analysis of test samples taken indicated that measured concentrations of the test item remained within 80-120% of the nominal concentrations and were maintained over the 72h test period (i.e. deviation <20%). The stability of the substance was thus confirmed over the test period. The results (growth rate) demonstrated that algal growth was significantly inhibited at all tested concentration. The definitive test conditions were based on these results.

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. Microscopic observation confirmed that the algae appeared "normal" at the end of the test except at the highest concentration: algae cells were found to be inflated.
Chemical analysis of test samples taken indicated that measured concentrations of the test item remained within 80-120% of the nominal concentrations and were maintained over the 72h test period (i.e. deviation <20%). Chemical analysis of the test item showed the stability of the substance over the 72-hour test period, the exposure concentrations were based on the nominal concentrations:

-72h-ErC50 = 3.5 mg/L
-72h-ErC10 = 1.87 mg/L

Results with reference substance (positive control):

The sensitivity of the test system and the methodology are evaluated every two months by performing an algal growth inhibition test on potassium dichromate. The latest value of ErC50 obtained in January 2018 was 0.92 mg/L. For information, according to ISO 8692 standard, 72h-ErC50 obtained from an inter-laboratory exercise on potassium dichromate was in the range 0.65 to 1.73 mg/L.

Reported statistics and error estimates:

Statistical analysis was performed with the software ToxRat 3.2.1.

Table 1: Algal cell density throughout the definitive test.

Test solution	Replicate	Algal density at 24h (Fd, ¢ *10^4/ ml )			Algal density at 48h (Fd, ¢ *10^4/ ml )			Algal density at 72h (Fd, ¢ *10^4/ ml )			Average specific growth rate J0-J3				Specific growth rate inhibition (%)
		Replicate mean	Mean	RSD	Replicate mean	Mean	RSD	Replicate mean	Mean	RSD	Replicate mean	Average	Sx	CV
Control	a	6,75	4,74	21,59	23,61	23,29	7,13	153,56	133,48	12,45	1,678	1,629	0,042	2,588	Inhibition (per replicate)	Inhibition (mean)
	b	4,59			21,82			112,82			1,575
	c	4,30			26,29			144,76			1,658
	d	4,27			23,14			144,09			1,657
	e	3,86			21,71			116,33			1,585
	f	4,68			23,21			129,32			1,621
0,16	a	6,32	4,88	25,64	25,96	23,94	7,29	160,11	145,38	9,46	1,692	1,659	0,031	1,887	-3,9	-1,8
	b	4,30			22,91			143,18			1,655				-1,6
	c	4,03			22,96			132,85			1,630				0,0
0,32	a	4,73	4,14	16,46	22,71	22,83	2,56	126,90	121,48	3,90	1,614	1,600	0,013	0,805	0,9	1,8
	b	4,30			23,46			119,39			1,594				2,1
	c	3,39			22,31			118,14			1,591				2,4
0,64	a	4,57	4,08	11,00	23,81	24,39	2,17	86,63	101,16	12,60	1,487	1,537	0,044	2,836	8,7	5,7
	b	3,99			24,86			106,38			1,556				4,5
	c	3,69			24,51			110,45			1,568				3,7
1,24	a	4,17	3,58	14,61	29,25	25,23	13,97	84,10	83,07	2,21	1,477	1,473	0,007	0,502	9,3	9,6
	b	3,17			23,72			84,15			1,478				9,3
	c	3,40			22,70			80,95			1,465				10,1
2,48	a	3,54	3,64	5,89	18,08	16,72	12,54	48,66	41,22	17,18	1,295	1,236	0,057	4,616	20,5	24,1
	b	3,89			14,31			34,57			1,181				27,5
	c	3,50			17,77			40,44			1,233				24,3
5,00	a	3,17	3,05	3,51	6,22	5,08	21,44	3,31	2,89	13,44	0,399	0,351	0,044	12,538	75,5	78,4
	b	3,01			4,05			2,80			0,343				79,0
	c	2,97			4,98			2,55			0,312				80,8

Table 2: Measured pH and dissolved oxygen in the definitive test

Test solution (mg/L)	pH		Dissolved O2 (mg/L)
	0h	72h	0h	72h
Control	8.2	8.7	9.8	10.5
0.16	8.1	8.9	9.8	10.3
0.32	8.1	8.9	9.8	10.4
0.64	8.1	8.8	9.7	10.2
1.24	8.1	8.9	9.5	9.8
2.48	8.1	8.9	9.0	9.2
5.00	8.1	8.9	8.4	8.7

NA: Not Applicable

 

Validity criteria fulfilled:

yes

Conclusions:

Key aquatic toxicity figures of sec-butylamine to Pseudokirchneriella subcapitata in terms of growth rate are:
-72h-ErC50 = 3.5 mg/L
-72h-ErC10 = 1.87 mg/L

Executive summary:

This study was designed to determine the effects of the test item on the growth of Pseudokirchneriella subcapitata in a 72-hour test according to the OECD 201 Guideline. The tests solutions were prepared from dilutions of a stock solution. pH was adjusted where relevant to fall within pH 7-8 range. The results are reported as growth rate inhibitions. The concentrations that result in a 10 and 50% reduction in growth rate (72h-ErC10 and 72h-ErC50, respectively) were determined with Toxrat 3.2. Chemical analysis of test samples taken indicated that measured concentrations of the test item remained within 80-120% of the nominal concentrations and were maintained over the 72h test period (i.e. deviation <20%). Based on these results, the exposure concentrations were based on nominal concentrations:

-72h-ErC50 = 3.5 mg/L

-72h-ErC10 = 1.87 mg/L

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

§ The biomass in the control cultures increased exponentially by a factor of 133.5 within the 72-hour test period (required: at last 16-fold)

§ The mean coefficients of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures were less than or equal to 11.1 (required: < 35%)

§ The coefficient of variation of average specific growth rates during the whole test period in the control cultures was 2.6% (required: < 7%)

Description of key information

This study was designed to determine the effects of the test item on the growth ofPseudokirchneriella subcapitatain a 72-hour test according to the OECD 201 Guideline. The tests solutions were prepared from dilutions of a stock solution. pH was adjusted where relevant to fall within pH 7-8 range. The results are reported as growth rate inhibitions. The concentrations that result in a 10 and 50% reduction in growth rate (72h-ErC10 and 72h-ErC50, respectively) were determined with Toxrat 3.2. Chemical analysis of test samples taken indicated that measured concentrations of the test item remained within 80-120% of the nominal concentrations and were maintained over the 72h test period (i.e. deviation <20%). Based on these results, the exposure concentrations were based on nominal concentrations:

-72h-ErC50 = 3.5 mg/L

-72h-ErC10 = 1.87 mg/L

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

§ The biomass in the control cultures increased exponentially by a factor of 133.5 within the 72-hour test period (required: at last 16-fold)

§ The mean coefficients of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures were less than or equal to 11.1 (required: < 35%)

§ The coefficient of variation of average specific growth rates during the whole test period in the control cultures was 2.6% (required: < 7%)

Key value for chemical safety assessment

EC50 for freshwater algae:

3.5 mg/L

EC10 or NOEC for freshwater algae:

1.87 mg/L