2-methylpentane-1,5-diamine

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental phase: 2019-10-08 to 2019-11-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Commission Regulation (EC) 761/2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006, Annex 5 corrected: 28 July 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stabilized by the addition of 0.01 M ammonium formate and 0.1% formic acid solution and stored frozen prior to analysis. A set of duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

A nominal amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1 liter to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 10, 18, 32 and 56 mg/L. An aliquot (400 mL) of each of the stock solutions was separately inoculated with algal suspension (2.5 mL) to give an initial nominal cell density of 5.00 x 103 cells/mL. Prior to inoculation, the pH of all stock
solutions was checked and adjusted to 7.5 ± 1 ºC.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

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

Details on test organisms:

Raphidocelis subcapitata strain CCAP 278/4 (= Pseudokirchneriella subcapitata strain CCAP 278/4)
Origin: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland;

Inoculum culture:
Approximately 3 to 4 days before the start of the test, inoculum culture of algae was set up at an initial cell density of approximately 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 10^5 to 10^6 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Hardness:

Calcium & Magnesium: 0.09 mM

Test temperature:

24 ±1 °C

pH:

Start of the test (t= 0 h):
Control pH 7.9
Treatments pH 7.8-8.0

End of the study (t= 72 h):
Control pH 8.8
Treatments pH 8.2 (100 mg/L), 8.5 (56 mg/L), 8.7 (32 mg/L) and 8.8 (18 and 10 mg/L)

Dissolved oxygen:

Not applicable

Salinity:

n.a.

Conductivity:

not reported

Nominal and measured concentrations:

Nominal concentrations:
10, 18, 32, 56 and 100 mg/L

Measured concentrations:
t= 0 h [mg/L]: 8.24, 15.5, 29.1, 44.9, 98.6;
t= 72 h [mg/L]: 7.90, 15.1, 28.9, 49.7, 92.4;

Details on test conditions:

See section "Any other information on materials and methods incl. tables".

Reference substance (positive control):

yes

Remarks:

A positive control test using potassium dichromate as the reference item was performed twice in a 12-month period to demonstrate satisfactory conditions of the test.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

As analytically confirmed concentrations (0 h; 72 h) were very close to nominal, toxicity evaluation was based on nominal values.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

As analytically confirmed concentrations (0 h; 72 h) were very close to nominal, toxicity evaluation was based on nominal values.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

60 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

As analytically confirmed concentrations (0 h; 72 h) were very close to nominal, toxicity evaluation was based on nominal values.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 52 to 70 mg/L

Details on results:

As analytically confirmed concentrations (0 h; 72 h) were very close to nominal, toxicity evaluation was based on nominal values.
All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures.
At the start of the test, all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 10, 18, 32 and 56 mg/L test cultures were green dispersions (due to the presence of algal cells) whilst the 100 mg/L test cultures were slightly pale green dispersions.
For further information, see section "Any other information on results incl. tables".

Results with reference substance (positive control):

A positive control (Covance Study Number TP88DT) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L. The positive control was conducted between 16 July 2019 and 19 July 2019.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 to 72 hour) : 1.3 mg/L; 95% confidence limits 1.2 to 1.4 mg/L
EyC50 (0 to 72 hour) : 0.55 mg/L; 95% confidence limits 0.49 to 0.63 mg/L
No Observed Effect Concentration based on growth rate: 0.25 mg/L
No Observed Effect Concentration based on yield: 0.25 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration based on yield: 0.50 mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference item:
According to in-house data from the last 5 years the mean ErC50 value is 1.4 mg/L (standard deviation = 0.22) and the mean EyC50 value is 0.67 mg/L (standard deviation = 0.12).

Reported statistics and error estimates:

Statistical analysis of the growth rate data was carried out for the control and all test concentrations using Shapiro-Wilk’s Test on Normal Distribution, Levene’s Test on Variance Homogeneity followed by Dunnett’s Multiple t-test procedure. The normality check and variance homogeneity check were passed (p> 0.01) and the minimum detectable difference was -2.7% for Dunnett’s Multiple t-test Procedure.
Statistical analysis of the growth rate data was carried out for the control and all test concentrations using Shapiro-Wilk’s Test on Normal Distribution, Levene’s Test on Variance Homogeneity followed by Dunnett’s Multiple t-test procedure. The normality check and variance homogeneity check were passed (p> 0.01) and the minimum detectable difference was -12.5% for Dunnett’s Multiple t-test Procedure.

Range-finding Test
No effect on growth was observed at the test concentrations of 0.10, 1.0 and and 10 mg/L. However, growth was observed to be reduced at 100 mg/L.
Based on this information test concentrations of 10, 18, 32, 56 and 100 mg/L were selected for the definitive test.
Chemical analysis of the test preparations at 0 and 72 hours showed that near nominal measured concentrations were obtained indicating that the test item was stable under test conditions.

Definitive Test
Verification of Test Concentrations
Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to be near nominal except for the 10 mg/L test concentration at 72 hours where a measured concentration of 79% of nominal was obtained. Given that this was only just below the 80-120% threshold for which OECD TG 201 allows for evaluation based on nominal concentrations, and that no significant decline was observed over the test duration, it was considered acceptable to base the results on the nominal concentrations.
The following analytical results at 0 and 72 hours were obtained:

Analytical Identification	Sample Description	Time Point (Hours)	MDMP Detected (mg/L)	Nominal recovery (%)
19/5949	Control	0	ND	-
19/5950	10 mg/L		8.24	82
19/5951	18 mg/L		15.5	86
19/5952	32 mg/L		29.1	91
19/5953	56 mg/L		44.9	80
19/5954	100 mg/L		98.6	99
19/5955	Control	72	ND	-
19/5956	10 mg/L		7.90	79
19/5957	18 mg/L		15.1	84
19/5958	32 mg/L		28.9	90
19/5959	56 mg/L		49.7	89
19/5960	100 mg/L		92.4	92

ND – none detected less than the limit of detection of 2 mg/L

Growth Data
Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in following Table. Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks (R = Replicate).

Nominal Concentration (mg/L)		pH	Cell Densities (cells per mL)			pH
		0 Hours	24 Hours	48 Hours	72 Hours	72 Hours
Control	R1	7.9	3.43E+04	2.19E+05	1.47E+06	8.8
	R2		4.16E+04	1.93E+05	1.35E+06
	R3		3.51E+04	2.11E+05	1.32E+06
	R4		3.44E+04	1.91E+05	1.26E+06
	R5		3.43E+04	1.90E+05	1.29E+06
	R6		3.35E+04	2.14E+05	1.38E+06
	Mean		3.55E+04	2.03E+05	1.34E+06
10	R1	7.9	2.99E+04	1.84E+05	1.04E+06	8.8
	R2		2.63E+04	1.70E+05	1.23E+06
	R3		3.18E+04	2.03E+05	1.26E+06
	Mean		2.94E+04	1.86E+05	1.18E+06
18	R1	8.0	2.67E+04	1.64E+05	9.81E+05	8.8
	R2		2.45E+04	1.44E+05	9.45E+05
	R3		2.46E+04	1.46E+05	9.44E+05
	Mean		2.53E+04	1.52E+05	9.57E+05
32	R1	7.9	3.33E+04	1.82E+05	1.30E+06	8.7
	R2		2.58E+04	1.67E+05	1.11E+06
	R3		2.61E+04	1.60E+05	9.31E+05
	Mean		2.84E+04	1.70E+05	1.12E+06
56	R1	7.9	1.99E+04	1.43E+05	8.07E+05	8.5
	R2		1.71E+04	1.53E+05	7.43E+05
	R3		2.45E+04	1.42E+05	8.46E+05
	Mean		2.05E+04	1.46E+05	7.99E+05
100	R1	7.8	1.88E+04	1.14E+05	6.20E+05	8.2
	R2		1.47E+04	9.52E+04	5.79E+05
	R3		1.85E+04	1.21E+05	5.62E+05
	Mean		1.73E+04	1.10E+05	5.87E+05

Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in the following table (R = Replicate; SD = Standard Deviation; [ ] = Increase in growth compared to controls; - = Not applicable).

Nominal Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield
		0 to 72 Hour	% Inhibition	0 - 72 h	% Inhibition
Control	R1	0 079	-	1.46E+06	-
	R2	0 078		1.34E+06
	R3	0 077		1.31E+06
	R4	0 077		1.26E+06
	R5	0 077		1.28E+06
	R6	0 078		1.37E+06
	Mean	0 078		1.34E+06
	SD	0 001		7.21E+04
10	R1	0 074	5	1.04E+06	12
	R2	0 076	3	1.23E+06
	R3	0 077	1	1.26E+06
	Mean	0 076	3	1.17E+06
	SD	0 002		1.18E+05
18	R1	0 073	6	9.76E+05	29
	R2	0 073	6	9.40E+05
	R3	0 073	6	9.39E+05
	Mean	0 073	6	9.52E+05
	SD	0 000		2.08E+04
32	R1	0 077	1	1.30E+06	17
	R2	0 075	4	1.11E+06
	R3	0 073	6	9.26E+05
	Mean	0 075	4	1.11E+06
	SD	0 002		1.87E+05
56	R1	0 071	9	8.02E+05	41
	R2	0 069	12	7.38E+05
	R3	0 071	9	8.41E+05
	Mean	0 070	10	7.94E+05
	SD	0 001		5.18E+04
100	R1	0 067	14	6.15E+05	57
	R2	0 066	15	5.74E+05
	R3	0 066	15	5.57E+05
	Mean	0 066	15	5.82E+05
	SD	0 001		2.98E+04

The mean cell densities versus time for the definitive test are presented in Figure 1 (attached illustration). Percentage inhibition values are plotted against test concentration in Figure 2 and Figure 3 (attached illustration).
From the data given above, it is obvious that the growth rate (r) and yield (y) of Raphidocelis subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72-Hour exposure period.
Accordingly, the following results were determined from the data:

Inhibition of Growth Rate
ErC10 (0 to 72 hour) : 60 mg/L; 95% confidence limits 52 to 70 mg/L
ErC20 (0 to 72 hour) : >100 mg/L
ErC50 (0 to 72 hour) : >100 mg/L
where ErCx is the test concentration that reduced growth rate by x%. Goodness of fit (P(Chi^2)) was calculated to be 1.000, whilst the significance of the dose response relationship (p(F)) was <0.001.

There were no statistically significant differences (P>=0.05), between the control and the 10 mg/L test concentration; however, all other test concentrations were significantly different (P<0.05) and, therefore the No Observed Effect Concentration (NOEC) based on growth rate was 10 mg/L. Correspondingly the Lowest Observed Effect Concentration (LOEC) based on growth rate was 18 mg/L.

Inhibition of Yield

EyC10 (0 to 72 hour) : Could not be determined
EyC20 (0 to 72 hour) : 19 mg/L; 95% confidence limits 13 to 23 mg/L
EyC50 (0 to 72 hour) : 87 mg/L; 95% confidence limits 71 to 116 mg/L
where EyCx is the test concentration that reduced growth rate by x%. Goodness of fit (P(Chi^2)) was calculated to be 1.000, whilst the significance of the dose response relationship (p(F)) was <0.001.

All test concentrations were found to be significantly different (P<0.05) from the control, therefore no NOEC could be determined.

Validity criteria fulfilled:

yes

Conclusions:

The following results in relation to growth rate were obtained from an algal growth inhibition test (OECD TG 201, GLP) based on analytically confirmed nominal test concentrations:
ErC10 (0 to 72 hour) : 60 mg/L; 95% confidence limits 52 to 70 mg/L
ErC20 (0 to 72 hour) : >100 mg/L
ErC50 (0 to 72 hour) : >100 mg/L
NOEC (growth rate; 0 to 72 hour): 10 mg/L

Executive summary:

A study on green algae growth inhibition with the test item MPMD (2-methylpentane-1,5-diamine) was performed compliant with GLP according to OECD TG 201 (2006) and EU method C.3 (2009) using Raphidocelis subcapitata (= Pseudokirchneriella subcapitata).
Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to aqueous solutions of the test item at concentrations of 10, 18, 32, 56 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C. Due to the caustic nature of the test item, the pH of the stock solution was adjusted to pH 7.5 with HCl before use in the test.
Samples of the algal populations were removed daily, and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.
Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to be near nominal and so the results are based on nominal test item concentrations.
All validity criteria of the testing guidelines were met.

The following results were obtained for the period of 0h to 72 hours based on the analytically confirmed nominal test concentrations:
Inhibition of Growth Rate
ErC10 (0 to 72 hour) : 60 mg/L; 95% confidence limits 52 to 70 mg/L
ErC20 (0 to 72 hour) : >100 mg/L
ErC50 (0 to 72 hour) : >100 mg/L
NOEC (growth rate; 0 to 72 hour): 10 mg/L

Inhibition of Yield
EyC10 (0 to 72 hour) : Could not be determined
EyC20 (0 to 72 hour) : 19 mg/L; 95% confidence limits 13 to 23 mg/L
EyC50 (0 to 72 hour) : 87 mg/L; 95% confidence limits 71 to 116 mg/L
NOEC (yield; 0 to 72 hour): Could not be determined

Description of key information

The following results in relation to growth rate were obtained from an algal growth inhibition test (OECD TG 201, GLP) based on analytically confirmed nominal test concentrations:
ErC10 (0 to 72 hour) : 60 mg/L; 95% confidence limits 52 to 70 mg/L
ErC20 (0 to 72 hour) : >100 mg/L
ErC50 (0 to 72 hour) : >100 mg/L
NOEC (growth rate; 0 to 72 hour): 10 mg/L

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

60 mg/L

Additional information

A study on green algae growth inhibition with the test item MPMD (2-methylpentane-1,5-diamine) was performed compliant with GLP according to OECD TG 201 (2006) and EU method C.3 (2009) using Raphidocelis subcapitata (= Pseudokirchneriella subcapitata).
Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to aqueous solutions of the test item at concentrations of 10, 18, 32, 56 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C. Due to the caustic nature of the test item, the pH of the stock solution was adjusted to pH 7.5 with HCl before use in the test.
Samples of the algal populations were removed daily, and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.
Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to be near nominal and so the results are based on nominal test item concentrations.
All validity criteria of the testing guidelines were met.

The following results were obtained for the period of 0h to 72 hours based on the analytically confirmed nominal test concentrations:
Inhibition of Growth Rate
ErC10 (0 to 72 hour) : 60 mg/L; 95% confidence limits 52 to 70 mg/L
ErC20 (0 to 72 hour) : >100 mg/L
ErC50 (0 to 72 hour) : >100 mg/L
NOEC (growth rate; 0 to 72 hour): 10 mg/L

Inhibition of Yield
EyC10 (0 to 72 hour) : Could not be determined
EyC20 (0 to 72 hour) : 19 mg/L; 95% confidence limits 13 to 23 mg/L
EyC50 (0 to 72 hour) : 87 mg/L; 95% confidence limits 71 to 116 mg/L
NOEC (yield; 0 to 72 hour): Could not be determined