Reaction products of alcohols, C11-14-iso, C13 rich and phosphorus pentoxide

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:

from 2018-05-25 to 2018-05-31, with the definitive exposure phase from 2018-05-28 to 2018-05-31 at the test facility.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Principles of method if other than guideline:

None

GLP compliance:

yes

Specific details on test material used for the study:

Test item N-(Oleyl alkyl)- 1,3-propanediamine mono oleate (100%)

CAS no 40027-38-1

Batch no. P15-005

Purity (certified) 99.62% (100% - water content)
0.38% (water content)

Water solubility 80 mg/L (CMC acc. to OECD 115)

Appearance Beige molten (paste-like)

Stability under test not specified
conditions

Best before date 2020-02-18

Recommended storage Keep container tightly closed in a dry and well ventilated place. Avoid elevated temperatures.

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
All concentration levels and the control were analytically verified via LC-MS/MS after 0 (start of exposure) and 72 hours (end of the exposure) with algae.
The samples were analysed with an LC-MS/MS method, implemented under non-GLP and documented in the GLP raw data. The method was validated.

Sampling for the analytical monitoring
At the start of the exposure (0 hours), samples of the fresh media were taken after preparation of the test concentrations and analyzed.
At the end of the exposure (72 hours), samples of the old media were taken from the test vessels for analysis.

Vehicle:

no

Details on test solutions:

Preparation of the saturated solutions
A stock solution with an nominal loading of 100 mg/L was prepared with dilution water as specified in Table 2 three days before the start of the exposure. A glass bottle was filled with an appropriate amount of the dilution water, an appropriate volume of the test item (density: 0.98 g/cm³ (20 °C) was taken into account) was placed by pipette onto the water surface and the bottle was closed with the screw cap. A slow stirring procedure was applied. Gentle stirring (to avoid formation of an emulsion) was carried out for 72 hours with a magnetic stirrer at room temperature. Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL). The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL of the filtrate were discarded. The filtration was interrupted for 15 minutes to allow adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. The following solution was used as the highest concentration in the test and for preparation the dilution levels. During filtration, the filter was always be kept covered. The solution was checked via laser beam (Tyndall effect) for undissolved test item.

Test concentrations
Five concentrations in a geometric series with a factor of √10, prepared by dilution of the stock solution 100 mg/L with dilution water, were tested as
follows: 1.00 – 3.16 – 10.0 – 31.62 – 100 % of the saturated solution.
The dilution levels are based on the results of a preliminary range finding test (non-GLP).

Control Six replicates (without test item) were tested under the same test conditions as the test item replicates.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDÁK
- Strain: CCAP
- Source (laboratory, culture collection):
Culture Collection of Algae and Protozoa (CCAP)
SAMS Research Services Ltd
Dunstaffnage Marine Laboratory
Dunbeg, OBAN; Argyll PA37 1QA; Scotland, UK

- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 2590 – 5180 lux corresponding to 35 - 70 µE ∙ m-2 ∙ s-1 for 24 h per day.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Hardness:

Not specified

Test temperature:

min.: 22.0 max.: 23.0 mean value: 22.5

pH:

Calculated test item concentration pH-values
[mg/L] Start; 0 hours End; 72 hours
2.89 7.41 7.53
0.913 7.51 7.73
0.289 7.57 8.00
0.0913 7.68 8.03
0.0289 7.70 8.34
Control 8.15 8.47

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: Nominal: approximately 2 - 5 x 10^3 cells/mL, Actual: mean 6442 cells/mL
- Control end cells density: Mean 1558142 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Composition of Dilution Water
according to the guideline
Component Concentration [mg/L]
NH4Cl 15
MgCl2 x 6 H2O 12
CaCl2 x 2 H2O 18
MgSO4 x 7 H2O 15
KH2PO4 1.6
FeCl3 x 6 H2O 0.064a2EDTA x 2 H2O 0.1
H3BO3 0.185
MnCl2 x 4 H2O 0.415
ZnCl2 3 x 10^-3
Na2MoO4 x 2 H2O 7 x 10^-3
CoCl2 x 6 H2O 1.5 x 10^-3
CuCl2 x 2 H2O 1 x 10^-5
NaHCO3 50
pH 8.1 ± 0.2



OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120
µE ∙ m-2 ∙ s-1, mean value: 6003 lux,measured: 5244 - 6535 with a mean of 5560 lux



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
-Chlorophyll a-fluorescence
The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was found in the definitive test in the saturated solution.

Reference substance (positive control):

yes

Remarks:

(Potassium dichromate)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.289 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

other: growth rate und yield inhibition

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.913 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield inhibition

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.871 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI: (0.455 - 2.40)

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

1.17 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI: (0.974 - 2.47)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.9 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI: (1.23 - 2.68)

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.469 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

other: Yield inhibition

Remarks on result:

other: CI (0.341 - 0.600)

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.593 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

other: Yield inhibition

Remarks on result:

other: CI (0.456 - 0.700)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.963 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

other: Yield inhibition

Remarks on result:

other: CI: (0.870 - 1.11)

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: The test media were clear throughout the exposure phase.
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

The toxicity of potassium dichromate (SIGMA ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2018-03-26 to 2018-03-29. The reference item toxicity is in the valid range following test facility SOPS.

EC50-Values of the Reference Item
based on nominal concentrations mg/L, (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Growth Rate inhibition
ErC50 1.09 0.759 ± 0.555
95% confidence interval 0.842 – 1.31
Yield inhibition
EyC50 0.548 0.401 ± 0.297
95% confidence interval 0.439 – 0.660
SD = Standard deviation

Reported statistics and error estimates:

Calculation of the test item Concentrations
For calculation of the test item concentrations, the geometric mean measured concentration of the stock solution was determined. The other concentration levels were calculated based on this geometric mean measured stock solution concentration with the dilution factor (√10). This procedure was carried out, because the measured concentrations of the three lowest concentration levels were below the LOQ and all concentration levels were diluted from the same stock solution.

EC-values and statistical analyses
EC10-, EC20- and EC50-values with confidence intervals of growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression.

NOEC, LOEC and statistical analyses
The NOEC and LOEC were determined by calculation of significant differences of growth rates and yield. As a standard, One Way Analysis of Variance (ANOVA) and DUNNETT’s test were used for NOEL/LOEL calculations. When running a One Way Analysis of Variance, a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05. As the Normality Test failed for growth rate, the data was square-transformed for analysis.

Software
The data for the tables in this report were computer-generated and have been rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures.
Calculations were carried out using software
• Excel, MICROSOFT CORPORATION
• SigmaPlot, SPSS INC.
• GraphPad Prism, GRAPHPAD SOFTWARE, INC.

Cell Densities

Calculated test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 hours		24 hours		48 hours		72 hours
2.89		1		6442		9140		11035		10133
		2		6442		10287		14637		23604
		3		6442		11094		10665		11454
		Mean		6442		10174		12112		15064
0.913		1		6442		26019		125528		842603
		2		6442		26041		151673		805762
		3		6442		19577		131525		869016
		Mean		6442		23879		136242		839127
0.289		1		6442		31309		218482		1644549
		2		6442		31265		204352		1668133
		3		6442		25302		185594		1408414
		Mean		6442		29292		202809		1573699
0.0913		1		6442		32096		244856		1634123
		2		6442		31875		214560		1660288
		3		6442		32310		141217		1824680
		Mean		6442		32094		200211		1706364
0.0289		1		6442		27717		225910		1604482
		2		6442		31714		204898		1598921
		3		6442		33136		236575		1711130
		Mean		6442		30856		222461		1638178
Control		1		6442		32056		256186		1722649
		2		6442		32590		248868		1626775
		3		6442		26195		207281		1517148
		4		6442		31361		191185		1308419
		5		6442		32876		251460		1509402
		6		6442		31649		273544		1664459
		Mean		6442		31121		238087		1558142

Evaluation after 72 hours

             Statistically significant differences of growth rates and yield compared to
                  control values are marked (+), not significant differences are marked (-).

 

Calculated test item concentration	Replicate	Growth Rate		Inhibition of Growth Rate	Yield		Inhibition of Yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
2.89	1		0.15	92		3691	100
	2		0.43	76		17162	99
	3		0.19	90		5012	100
	Mean	(+)	0.26	86	(+)	8622	99
0.913	1		1.63	11		836161	46
	2		1.61	12		799320	48
	3		1.64	11		862574	44
	Mean	(+)	1.62	11	(+)	832685	46
0.289	1		1.85	-1		1638107	-6
	2		1.85	-1		1661691	-7
	3		1.80	2		1401972	10
	Mean	(-)	1.83	0	(-)	1567257	-1
0.0913	1		1.85	-1		1627681	-5
	2		1.85	-1		1653846	-7
	3		1.88	-3		1818238	-17
	Mean	(-)	1.86	-2	(-)	1699922	-10
0.0289	1		1.84	-1		1598040	-3
	2		1.84	-1		1592479	-3
	3		1.86	-2		1704688	-10
	Mean	(-)	1.85	-1	(-)	1631736	-5
Control	1		1.86			1716207
	2		1.84			1620333
	3		1.82			1510706
	4		1.77			1301977
	5		1.82			1502960
	6		1.85			1658017
	Mean		1.83			1551700

 

 

Section-by-Section and Average Specific Growth Ratesof the Control Group
(0 - 72 hours)

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 hours)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.61	2.08	1.91	1.86	0.240	12.9	13.4
	2	1.62	2.03	1.88	1.84	0.208	11.3
	3	1.40	2.07	1.99	1.82	0.364	20.0
	4	1.58	1.81	1.92	1.77	0.173	9.78
	5	1.63	2.04	1.79	1.82	0.204	11.2
	6	1.59	2.16	1.81	1.85	0.285	15.4
				Mean	1.83
				SD ±	0.03
				CV [%]	1.79

 

SD = Standard deviation    CV = Coefficient of variation

Validity criteria fulfilled:

yes

Conclusions:

In this study, the effect values of Reaction products of alcohols, C11-14-iso,C13 rich and phosphorus pentoxide on the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours were determined as follows (based on calculated test item concentrations):
The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 1.90 (1.23 – 2.68) mg/L and 0.963 (0.870 – 1.11) mg/L, respectively. The NOEC for both growth rate and yield was 0.289 mg/L, respectively.

Executive summary:

The toxicity of Reaction productsof alcohols, C11-14-iso,C13 rich and phosphorus pentoxide o the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3 from 2018-05-25 to2018-05-31, with the definitive exposure phase from 2018-05-28 to 2018-05-31 at the test facility.The aim of the study was the determination of the effects on growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 6442 cells/mL.Five concentrations were tested in a geometrical series with factor of√10 in the concentration range of 1.00 - 3.16 - 10.0 - 31.6 - 100% of the saturated solution. A saturated solution was prepared and slowly stirred for 72 hours. The solution was then filtrated and used for the preparation of the dilution solutions.

Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits. The measured concentrations in the three highest test solutions at the start of the exposure were 2.89 mg/L, 0.766 mg/L and 0.186 mg/L. All other test item solutions were < LOQ. At the end of exposure after 72 hours, the measured concentrations in the two highest concentrations were 104 and 88% of the initially measured test item concentrations, all other measurements were < LOQ. As the concentrations remained in the nominal range of the initially measured concentrations, all solutions were prepared from the saturated solution and the dilution factor was reflected in the measured concentrations, the exposure concentrations were calculated from the value for the saturated solution.

All effect values given are based on the calculated test item concentrations of Reaction products of alcohols, C11-14-iso,C13 rich and phosphorus pentoxide.

 

NOEC, LOEC, EC_x-values ofReactionproductsof alcohols, C11-14-iso,C13 rich and phosphorus pentoxide(0 - 72 hours)

                  (based on the calculated test item concentrations, with 95% confidence intervals)

	Growth Rate Inhibition calculated test item concentrations, with 95% confidence intervals [mg/L]
NOEC	0.289
LOEC	0.913
ErC10	0.871 (0.455 – 2.40)
ErC20	1.17 (0.974 – 2.47)
ErC50	1.90 (1.23 – 2.68)
	Yield Inhibition calculated test item concentrations, with 95% confidence intervals [mg/L]
NOEC	0.289
LOEC	0.913
EyC10	0.469 (0.341 – 0.600)
EyC20	0.593 (0.456 – 0.700)
EyC50	0.963 (0.870 – 1.11)

Description of key information

In this study, the effect values of Reaction products of alcohols, C11-14-iso,C13 rich and phosphorus pentoxide on the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours were determined as follows (based on calculated test item concentrations):

The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 1.90 (1.23 – 2.68) mg/L and 0.963 (0.870 – 1.11) mg/L, respectively. The NOEC for both growth rate and yield was 0.289 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

1.9 mg/L

EC10 or NOEC for freshwater algae:

0.8 mg/L