Fatty acids, C18-unsatd., compds. with diisopropanolamine N-C12-18-alkyl derivs.

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-01-05 to 2015-01-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study, no deficiencies

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C18-unsatd., compd. with 2-propanol, 1,1’-iminobis-, N-C12-18-(even numbered) alkyl derivs.

Cas Number:

2156594-85-1

Molecular formula:

Not applicable

IUPAC Name:

Fatty acids, C18-unsatd., compd. with 2-propanol, 1,1’-iminobis-, N-C12-18-(even numbered) alkyl derivs.

Test material form:

other: viscous liquid

Details on test material:

- Name of test material (as cited in study report): Fatty acids, C18-unsatd., compd. with 2-propanol, 1,1’-iminobis-, N-C12-18-(even numbered) alkyl derivs.
- Physical state: Brown yellow, slightly turbid, viscous liquid
- Storage condition of test material: Room temperature, protected from light, in original container

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 5 concentrations were tested in a geometrical series with a
dilution factor of 4√10: 0.100 - 0.178 - 0.316 - 0.562 - 1.00 mg/L, corresponding to geometric mean measured test item concentrations of 0.0518 - 0.0901 - 0.150 - 0.279 - 0.537 mg/L.

- Sampling method: Analytical evaluation of the test concentrations of Fatty acids, C18-unsatd., compd. with 2-propanol, 1,1’-iminobis-, N-C12-18-(even numbered) alkyl derivs. and the control were carried out via LC-MS/MS from freshly prepared media after 0 hours and old test media after 72 hours. Separate replicates for the test item analysis at the beginning of the exposure were prepared without algae. For the test item analysis after 72 hours, samples were taken out of the test replicates.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stocksolution 1 mg/L, freshly prepared with dilution water
Application Application was carried out by adding appropriate volumes of the stock solution to the replicate test vessels.
- Differential loading: 5 concentrations were tested in a geometrical series with a
dilution factor of 4√10: 0.100 - 0.178 - 0.316 - 0.562 - 1.00 mg/L, corresponding to geometric mean measured test item concentrations of 0.0518 - 0.0901 - 0.150 - 0.279 - 0.537 mg/L.
- Dilution water: According to the guideline, composition of dilution water see below -Controls: Six replicates (without test item) were tested under the same test conditions as the test replicates.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDÁK
- Strain: SAG 61.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Fresh stocks were prepared from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 hours per day.

ACCLIMATION
- Culturing media and conditions (same as test or not): Not the same, standard growth medium
- Any deformed or abnormal cells observed: No

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Test conditions

Hardness:

Not measured

Test temperature:

Mean: 22.5 °C, Min: 22.0 °C, Max: 23.0 °C

pH:

Geometric mean measured
test item concentration pH-value
[mg/L] Start; 0 h End; 72 h
0.537 7.94 7.91
0.279 7.96 7.98
0.150 7.99 8.10
0.0901 8.05 8.20
0.0518 8.09 8.26
Control 8.08 8.20

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to information on materials and methods

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks (sterile)
- Type (delete if not applicable): sealed with cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Agitation: 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: 7085 cells/mL
- Control end cells density: Mean 1731958 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None

GROWTH MEDIUM
- Standard medium used: Yes


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to the guideline (OECD)

Composition of Dilution water (OECD)

Component Concentration [mg/L]
NH4Cl 15
MgCl2  6 H2O 12
CaCl2  2 H2O 18
MgSO4  7 H2O 15
KH2PO4 1.6
FeCl3  6 H2O 0.064
Na2EDTA  2 H2O 0.1
H3BO3 0.185
MnCl2  4 H2O 0.415
ZnCl2 3 x 10-3
Na2MoO4  2 H2O 7 x 10-3
CoCl2  6 H2O 1.5 x 10-3
CuCl2  2 H2O 1 x 10-5
NaHCO3 50
pH-value 8.1  0.2

This medium had a nominal hardness of 0.24 mmol Ca+Mg/L.


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1 (nominal), 74.5 µE x m-2 x s-1 (measured, n = 30, CV = 7.38%)



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Fluorimeter, via
Chlorophyll-a fluorescence (excitation at 436 nm, emission at 685 nm), cell concentrations were related to all cell density values acc. to a calibration curve.



TEST CONCENTRATIONS
- Range finding study: Yes
Results of the Range Finding Test (0 – 72 h)

Saturated Solution [%] Specific Growth Rate Inhibition [%] Yield Inhibition [%]
100 100 100
10 100 100
1 100 100
0.1 -1 -6
negative inhibitions = increase of growth

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.353 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 0.325 - 0.401

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.205 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 0.172 - 0.230

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.15 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.279 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: Microscopic evaluation of the cells at start of the incubation period revealed no morphological abnormalities. At the end of the incubation period, the algae of the geometric mean measured concentration levels 0.150, 0.279 and 0.537 mg/L were deformed. At concentration level 0.150 mg/L cells were partly rounder and at concentration level 0.279 and 0.357 mg/L all cells were rounder. The other concentrations and the control showed no morphological abnormalities. All effect values are given based on the geometric mean measured test item concentrations.
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.749 (CI 0.715 - 0.788) mg/L
Yield Inhibition: 0.282 (CI 0.239 - 0.318) mg/L

Reported statistics and error estimates:

EC-values and statistical analysis:

EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression. Calculation of the confidence intervals of EC10-, EC20- and EC50-values were carried out using standard procedures.


NOEC/LOEC values:
NOEC/LOEC were determined by calculation of statistical significance of growth rate and yield. As standard One Way Analysis of Variance (ANOVA) and DUNNETT’s test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. 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. The Normality and Equal Variance Test failed for growth rates. The highest concentration level with 88 – 100 % inhibition effects was excluded to pass the Normality and Equal Variance Test.

Any other information on results incl. tables

EC-values and 95 % Confidence Intervals ofFatty acids,C18-unsatd., compd. with 2-propanol, 1,1’-iminobis-, N-C12-18-(even numbered) alkyl derivs.(0-72 hours)

                  based on geometric mean measured test item concentrations [mg/L]

 

	Growth Rate Inhibition geometric mean measured test item concentration [mg/L]
NOEC	0.150
LOEC	0.279
ErC10	0.205 (0.172 – 0.230)
ErC20	0.251 (0.229 – 0.265)
ErC50	0.353 (0.325 – 0.401)
	Yield Inhibition geometric mean measured test item concentration [mg/L]
NOEC	0.150
LOEC	0.279
EyC10	0.143 (0.129 – 0.158)
EyC20	0.162 (0.148 – 0.176)
EyC50	0.211 (0.196 – 0.228)

Cell Densities

 

Geometric Mean Measured Test Item Concentration [mg/L]	Replicate	Cell Density [cells/mL]
	No.	0 hours	24 hours	48 hours	72 hours
0.537	1	7085	10046	8300	3493
	2	7085	12102	13154	9044
	3	7085	12842	17430	13752
	Mean	7085	11663	12961	8763
0.279	1	7085	18227	87744	339802
	2	7085	18416	83090	332060
	3	7085	19824	109816	480305
	Mean	7085	18822	93550	384056
0.150	1	7085	21849	203576	1501882
	2	7085	22254	183297	1413570
	3	7085	25743	220123	1500222
	Mean	7085	23282	202332	1471891
0.0901	1	7085	23748	221278	1816087
	2	7085	24897	254325	2086135
	3	7085	23984	230129	1900282
	Mean	7085	24210	235244	1934168
0.0518	1	7085	27895	267353	1835409
	2	7085	20485	184327	1737403
	3	7085	24402	237433	1818543
	Mean	7085	24261	229704	1797118
Control	1	7085	25079	215979	1781559
	2	7085	20637	177906	1421471
	3	7085	22596	221869	1617551
	4	7085	25003	179127	1653606
	5	7085	28509	260859	2094037
	6	7085	24933	234810	1823523
	Mean	7085	24460	215092	1731958

Evaluation after 72 hours

Statistically significant differences of growth rates and yield compared to

control values are marked (+), not significant differences are marked (-).

Geometric Mean Measured Test Item Concentration	Replicate	Growth Rate		Inhibition of Growth Rate	Yield		Inhibition of Yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
0.537	1		-0.236	100		-3592	100
	2		0.081	96		1959	100
	3		0.221	88		6667	100
	Mean	(*)	0.022	95	(+)	1678	100
0.279	1		1.29	30		332717	81
	2		1.28	30		324975	81
	3		1.41	23		473220	73
	Mean	(+)	1.33	28	(+)	376971	78
0.150	1		1.79	2		1494797	13
	2		1.77	4		1406485	18
	3		1.79	2		1493137	13
	Mean	(-)	1.78	3	(-)	1464806	15
0.0901	1		1.85	-1		1809002	-5
	2		1.90	-4		2079050	-21
	3		1.86	-2		1893197	-10
	Mean	(-)	1.87	-2	(-)	1927083	-12
0.0518	1		1.85	-1		1828324	-6
	2		1.83	0		1730318	0
	3		1.85	-1		1811458	-5
	Mean	(-)	1.85	-1	(-)	1790033	-4
Control	1		1.84			1774474
	2		1.77			1414386
	3		1.81			1610466
	4		1.82			1646521
	5		1.90			2086952
	6		1.85			1816438
	Mean		1.83			1724873

 

negative inhibitions = increase of growth

* = excluded to pass Normality and Equal Variance Test

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study Fatty acids, C18-unsatd., compd. with 2-propanol, 1,1’-iminobis-, N-C12-18-(even numbered) alkyl derivs. was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (geometric mean measured test item concentrations): The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 0.353 (0.325 – 0.401) mg/L and 0.211 (0.196 – 0.228) mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 0.150 mg/L.

Executive summary:

The toxicity ofFatty acids,C18-unsatd., compd. with 2-propanol, 1,1’-iminobis-, N-C12-18-(even numbered) alkyl derivs.(batch number:DEG4273438)to the unicellular freshwater green algaPseudokirchneriella subcapitatawas determined according to the principles of OECD 201 andCouncil Regulation (EC) No. 440/2008/ Method C.3 from2015-01-05 to 2015-01-13with an exposure phase from 2015-01-05 to 2015-01-08atDr.U.Noack-Laboratorienin 31157 Sarstedt, Germany.The aim of the study was to assess the effects on the growth rate over a period of 72 hours.

 

The study was conducted under static conditions with an initial cell density of 7085 cells/ mL. Based on a preliminary range finding test, fiveconcentration levels were tested in a geometrical series with a dilution factor of⁴√10(nominal): 0.100 - 0.178 - 0.316 - 0.562 - 1.00 mg/L, corresponding to geometric mean measured test item concentrations of0.0518 - 0.0901 - 0.150 - 0.279 - 0.537 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditionswere within the acceptable limits.

 

All test concentrations ofFatty acids,C18-unsatd., compd. with 2-propanol, 1,1’-iminobis-, N-C12-18-(even numbered) alkyl derivs.and the control were analytically verified by LC-MS/MS at the start and the end of the exposure. Details of the analytical method are presented in section13. The analytical results are presented inTable9. The measured concentrations ofFatty acids,C18-unsatd., compd. with 2-propanol, 1,1’-iminobis-, N-C12-18-(even numbered) alkyl derivs.at the start of the exposure were in the range of 66 – 71 % of the nominal test item concentrations and 33 – 44 % at the end of the exposure.

 

All effect values are based on the geometric mean measured concentrations of the test item
Fatty acids,C18-unsatd., compd. with 2-propanol, 1,1’-iminobis-, N-C12-18-(even numbered) alkyl derivs..

 

EC-values and 95 % Confidence Intervals ofFatty acids,C18-unsatd., compd. with 2-propanol, 1,1’-iminobis-, N-C12-18-(even numbered) alkyl derivs.(0-72 hours)

                  based on geometric mean measured test item concentrations [mg/L]

 

	Growth Rate Inhibition geometric mean measured test item concentration [mg/L]
NOEC	0.150
LOEC	0.279
ErC10	0.205 (0.172 – 0.230)
ErC20	0.251 (0.229 – 0.265)
ErC50	0.353 (0.325 – 0.401)
	Yield Inhibition geometric mean measured test item concentration [mg/L]
NOEC	0.150
LOEC	0.279
EyC10	0.143 (0.129 – 0.158)
EyC20	0.162 (0.148 – 0.176)
EyC50	0.211 (0.196 – 0.228)