3-butoxypropylamine

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:

04 July 2017 - 01 September 2017

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)

Version / remarks:

2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

EC No. 761/2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Identification: BOPA
- CAS Number: 16499-88-0
- Physical state/Appearance: clear colorless liquid
- Batch: PFW160573
- Purity: 99.39%
- Expiry Date of the lot/batch: 26 September 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage Conditions: Room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
* Range-Finding Test: nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L
* Definitive test: 1.0, 3.2, 10, 32 and 100 mg/L.
- Sampling method:
* Range-Finding Test: A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis..
* Definitive Test: The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
* Range-Finding Tests
The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L for a period of 72 hours. The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were used for each control and test concentration. The test item was dissolved directly in culture medium. A nominal amount of test item (50 mg) was dissolved in culture medium and the volume adjusted to 500 mL to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 10, 1.0 and 0.10 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (4.6 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/L. The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity
* Definitive Test
A nominal amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1 litre to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 32, 10, 3.2 and 10 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (3.8 mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L.
* Verification of Test Concentrations
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 stored frozen prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4.
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 2”C. Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give 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 (100 to 150 rpm) and constant illumination at 24 ± 1”C until the algal cell density was approximately 10^4 to 10^5 cells/mL

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

not reported

Test temperature:

24 ± 1 °C
The temperature within the incubator was recorded daily.

pH:

control cultures: pH 7.8 at 0 hours to pH 8.3 at 72 hours.
A concentration dependent increase in pH was observed at the start of the test in the range of pH 7.8 at 1.0 mg/L through to pH 10.2 at 100 mg/L. This was considered to be due to an intrinsic property of the test item and as such no adjustment to the pH was made prior to the addition of the algal cells.

Dissolved oxygen:

Not reported

Salinity:

Not reported

Conductivity:

Not reported

Nominal and measured concentrations:

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 83% to 92% of nominal and so results are based on nominal test concentrations only.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): no
- Initial cells density: 5.90 x 10^5 cells per mL.
- Control end cells density: 5.00 x 10^3 cells per mL
- No. of vessels per concentration (replicates): three flasks each containing 100 mL were used for each treatment group.
- No. of vessels per control (replicates): Six flasks each containing 100 mL of test preparation were used for the control

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used:
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
NaNO3 25.5 mg/L
MgCl2.6H2O 12.16 mg/L
CaCl2.2H2O 4.41 mg/L
MgSO4.7H2O 14.6 mg/L
K2HPO4 1.044 mg/L
NaHCO3 15.0 mg/L
H3BO3 0.186 mg/L
MnCl2.4H2O 0.415 mg/L
ZnCl2 0.00327 mg/L
FeCl3.6H2O 0.160 mg/L
CoCl2.6H2O 0.00143 mg/L
Na2MoO4.2H2O 0.00726 mg/L
CuCl2.2H2O 0.000012 mg/L
Na2EDTA.2H2O 0.30 mg/L
The culture medium was prepared using reverse osmosis purified deionized water and the pH adjusted to 7.5 with 0.1N NaOH or HCl.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Light intensity and quality: continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: Samples were taken at 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00 x 10^3 cells/mL) was taken as the starting cell density. To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.
The data showed that the cell concentration of the control cultures increased by a factor of 126 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.
Nominal cell density of control at 0 hours: 5.00 x 10^3 cells per mL
Mean cell density of control at 72 hours : 6.28 x 10^6 cells per mL
The mean coefficient of variation for section by section specific growth rate for the control cultures was 13% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.
The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 3% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: 1.0, 3.2, 10, 32 and 100 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

14 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

It was not possible to calculate 95% confidence limits for the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits.

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- ErC10 (0 - 72 h): 9.8 mg/L
- LOErC (0 - 72 h): 10 mg/L
- NOEyC (0 - 72 h): 3.2 mg/L
- LOErC (0 - 72 h): 10 mg/L
- EyC10 (0 - 72 h): 7.1 mg/L
- EyC20 (0 - 72 h): 8.0 mg/L
- EyC50 (0 - 72 h):11 mg/L; 95% confidence limits 9.6 - 12 mg/L

Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 126 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.
Nominal cell density of control at 0 hours: 5.00 x 10^3 cells per mL
Mean cell density of control at 72 hours : 6.28 x 10^6 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 13% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.
The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 3% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Observations on Cultures
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 1.0, 3.2 and 10 mg/L, however no intact cells were observed to be present in the test cultures at 32 and 100 mg/L.

Water Quality Criteria
Temperature was maintained at 24 ± 1 ºC throughout the test.
The pH value of the control cultures (see Table 2) was observed to increase from pH 7.8 at 0 hours to pH 8.3 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.
A concentration dependent increase in pH was observed at the start of the test in the range of pH 7.8 at 1.0 mg/L through to pH 10.2 at 100 mg/L. This was considered to be due to an intrinsic property of the test item and as such no adjustment to the pH was made prior to the addition of the algal cells.

Observations on Test Item Solubility
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, 1.0 and 3.2 mg/L test cultures were observed to be green dispersions. The 10 mg/L test cultures were observed to be pale green dispersions whilst the 32 and 100 mg/L test cultures were observed to be clear colorless solutions.

Results with reference substance (positive control):

- Results with reference substance valid? yes
ErC50 (0 – 72 h): 1.4 mg/L; 95% confidence limits 1.2 – 1.5 mg/L
EyC50 (0 – 72 h): 0.60 mg/L; 95% confidence limits 0.52 – 0.69 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Reported statistics and error estimates:

Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). There were no statistically significant differences (P>0.05), between the control, 1.0 and 3.2 mg/L test concentrations however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 3.2 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 10 mg/L.

Table 2       Cell Densities and Percentage Inhibition of Growth from the Range-finding Test   

Nominal Concentration (mg/L)		Cell Densities* (cells per mL)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	4.27E+03	1.10E+06	-	-
	R2	5.55E+03	1.15E+06
	Mean	4.91E+03	1.13E+06
0.10	R1	5.07E+03	1.30E+06	[3]	[22]
	R2	5.63E+03	1.46E+06
	Mean	5.35E+03	1.38E+06
1.0	R1	4.70E+03	1.36E+06	[3]	[9]
	R2	4.85E+03	1.10E+06
	Mean	4.78E+03	1.23E+06
10	R1	4.75E+03	9.73E+05	4	24
	R2	4.61E+03	7.41E+05
	Mean	4.68E+03	8.57E+05
100	R1	4.05E+03	2.89E+04	73	99
	R2	5.10E+03	9.06E+03
	Mean	4.58E+03	1.90E+04

*    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.

R₁and R₂= Replicates 1 and 2

Table 3            Cell Densities and pH Values in the Definitive Test

Nominal Concentration (mg/L)		pH	Cell Densities* (cells per mL)			pH
		0 h	24 h	48 h	72 h	72 h
Control	R1	7.8	1.58E+04	1.29E+05	5.11E+05	8.3
	R2		2.27E+04	1.23E+05	8.06E+05
	R3		2.15E+04	1.10E+05	5.85E+05
	R4		2.40E+04	8.55E+04	5.78E+05
	R5		2.61E+04	1.38E+05	6.92E+05
	R6		2.68E+04	1.36E+05	5.97E+05
	Mean		2.28E+04	1.20E+05	6.28E+05
1.0	R1	7.8	2.61E+04	1.55E+05	7.64E+05	8.3
	R2		2.44E+04	1.23E+05	7.85E+05
	R3		2.57E+04	1.16E+05	7.46E+05
	Mean		2.54E+04	1.32E+05	7.65E+05
3.2	R1	7.9	1.96E+04	1.54E+05	7.38E+05	8.3
	R2		2.32E+04	1.21E+05	7.36E+05
	R3		1.92E+04	1.39E+05	7.41E+05
	Mean		2.07E+04	1.38E+05	7.38E+05
10	R1	8.6	2.20E+04	7.70E+04	4.53E+05	8.1
	R2		1.65E+04	6.43E+04	2.77E+05
	R3		1.39E+04	5.86E+04	3.45E+05
	Mean		1.75E+04	6.66E+04	3.58E+05
32	R1	9.5	1.07E+03	6.14E+03	7.36E+03	8.0
	R2		1.30E+03	1.70E+03	4.22E+03
	R3		1.50E+03	1.65E+03	4.61E+03
	Mean		1.29E+03	3.16E+03	5.40E+03
100	R1	10.2	1.70E+03	2.48E+03	1.26E+03	8.1
	R2		5.44E+02	1.09E+03	3.75E+03
	R3		5.12E+02	1.04E+03	2.73E+03
	Mean		9.17E+02	1.54E+03	2.58E+03

 

*    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.

R₁- R₆= Replicates 1 to 6

Table 4: Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.064		5.06E+05
	R2	0.071		8.01E+05
	R3	0.066		5.80E+05
	R4	0.066	-	5.73E+05	-
	R5	0.068		6.87E+05
	R6	0.066		5.92E+05
	Mean	0.067		6.23E+05
	SD	0.002		1.05E+05
1.0	R1	0.070	[4]	7.59E+05
	R2	0.070	[4]	7.80E+05
	R3	0.070	[4]	7.41E+05
	Mean	0.070	[4]	7.60E+05	[22]
	SD	0.000		1.97E+04
3.2	R1	0.069	[3]	7.33E+05
	R2	0.069	[3]	7.31E+05
	R3	0.069	[3]	7.36E+05
	Mean	0.069	[3]	7.33E+05	[18]
	SD	0.000		2.65E+03
10	R1	0.063	6	4.48E+05
	R2	0.056	16	2.72E+05
	R3	0.059	12	3.40E+05
	Mean	0.059	11	3.53E+05	43
	SD	0.004		8.85E+04
32	R1	0.005	93	2.36E+03
	R2	-0.002	103	-7.76E+02
	R3	-0.001	101	-3.95E+02
	Mean	0.001	99	3.97E+02	100
	SD	0.004		1.71E+03
100	R1	-0.019	128	-3.74E+03
	R2	-0.004	106	-1.25E+03
	R3	-0.008	112	-2.27E+03
	Mean	-0.010	115	-2.42E+03	100
	SD	0.008		1.25E+03

*       In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated

R₁-R₆= Replicates 1 to 6

SD= Standard Deviation

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period. Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 83% to 92% of nominal and so the results are based on nominal test concentrations only. Based on the growth rate, the EC50 was determined to be 14 mg/L and the NOEC was 3.2 mg/L. It was not possible to calculate 95% confidence limits for the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits.

Description of key information

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period (Vryenhoef H, 2018). Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 83% to 92% of nominal and so the results are based on nominal test concentrations only. Based on the growth rate, the EC50 was determined to be 14 mg/L and the NOEC was 3.2 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

14 mg/L

EC10 or NOEC for freshwater algae:

3.2 mg/L