Fatty acids, C16-18, reaction products with diethanolamine

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:

2013-03-11 to 2013-03-15

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The water accommodated fraction (WAF) was prepared with a nominal loading of the test item of 100 mg/L.
- Sampling method: The concentrations of the test item were analytically determined from all test concentrations and the control after 0 hours (test start) and 72 hours (test end). Samples at test start were taken from an additional replicate without algae. At the end of the test samples were taken from pooled replicate with algae.
- Sample storage conditions before analysis:All original samples were stored at room temperature until sample preparation, if necessary. Prepared samples were stored at room temperature until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The water accommodated fraction (WAF) was prepared with a nominal loading of the test item of 100 mg/L.
100 mg/L of the test item were weighed out and transferred with an appropriate amount of dilution water into a brown glass flask. This dispersion was shaken for 24 hours with 20 rpm at room temperature. After a separation phase of 1 hour, the aqueous phase or WAF was removed by siphoning (from the approximate centre of the glass flask) to give the 100 mg/L loading rate WAF.
- Eluate: Dilution water
- Differential loading: None, limit test
- Controls: Six replicates (without test item) were tested under the same test conditions as the test vessels.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 35 - 70 µE x m-2 x s-1 for 24 h per day.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No

Hardness:

0.24 mmol Ca+Mg/l

Test temperature:

min.: 22.0 max.: 23.0, mean value: 22.5 °C

pH:

Nominal test item loading pH-value
[mg/L] Start; 0 hours End; 72 hours
100 8.10 8.13
Control 8.14 8.29

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

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

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 103 cells/mL, Actual: mean 4332 cells/mL
- Control end cells density: Mean 631901 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  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 8.1  0.2




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, mean value: 67.9



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] fluorimeter, Cell density and self fluorescence was measured daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal.



TEST CONCENTRATIONS

Results of the Range Finding Test (0 - 72 hours)
Nominal Test Item Loading
[mg/L] Growth Rate Inhibition [%] Yield Inhibition [%]
100 1 3
10 1 3
1.0 -4 -22

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

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: No
- 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
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes

Current Study Valid Range (average ± 3 x SD)
Growth Rate Inhibition
ErC50 0.631 0.682 ± 0.452
95 %
confidence
interval 0.583 - 0.687
Yield Inhibition
EyC50 0.256 0.314 ± 0.144
95 %
confidence
interval 0.247 - 0.265
SD = Standard deviation

Reported statistics and error estimates:

NOEL/LOEL 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 NOEL/LOEl 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 Test failed for the growth rate, therefore the data were transformed (y = 10y).

Cell Densities

	Nominal test item loading		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 hours		24 hours	48 hours	72 hours
100		1		4332		18987	101569	505431
		2		4332		16644	87831	540623
		3		4332		18151	83375	499958
		4		4332		18456	96769	501908
		5		4332		16254	88662	500304
		6		4332		17890	90756	587924
		Mean		4332		17730	91494	522691
Control		1		4332		21516	128924	846474
		2		4332		20355	140510	737311
		3		4332		25205	155071	723662
		4		4332		24576	144812	698597
		5		4332		26051	143007	811565
		6		4332		22714	143171	693502
		Mean		4332		23403	142583	751852

Evaluation after 72 hours

                   Statistically significant differences of growth rates and yield compared to

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

Nominal test item loading	Replicate	Growth rate		Growth rate inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
100	1		1.59	8		501099	33
	2		1.61	6		536291	28
	3		1.58	8		495626	34
	4		1.58	8		497576	33
	5		1.58	9		495972	34
	6		1.64	5		583592	22
	Mean	(+)*	1.60	7	(+)	518359	31
Control	1		1.76			842142
	2		1.71			732979
	3		1.71			719330
	4		1.69			694265
	5		1.74			807233
	6		1.69			689170
	Mean		1.72			747520

Section-by-Section and Average Specific Growth Rates of the Control Group
                   (0 – 72 hours)

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 h)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.60	1.79	1.88	1.76	0.142	8.09	7.52
	2	1.55	1.93	1.66	1.71	0.198	11.6
	3	1.76	1.82	1.54	1.71	0.146	8.57
	4	1.74	1.77	1.57	1.69	0.106	6.27
	5	1.79	1.70	1.74	1.74	0.0461	2.65
	6	1.66	1.84	1.58	1.69	0.135	7.99
				Mean	1.72
				SD ±	0.03
				CV [%]	1.59

Validity criteria fulfilled:

yes

Conclusions:

In this study caused slightly inhibition on the growth rate and yield of the freshwater green alga Desmodesmus subspicatus was found after 72 hours exposure to the test item Fatty acids, C16-18-, reaction products with diethanolamine. All effect values given are based on the nominal loading level of the test item Fatty acids, C16-18-, reaction products with diethanolamine.

Executive summary:

The toxicity of Fatty acids, C16-18-, reaction products with diethanolamine to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 (2006) at Dr.U.Noack-Laboratorienin 31157 Sarstedt, Germany from 2013-03-11 to 2013-03-15, with the definitive exposure phase from 2013-03-12 to 2013-03-15. The aim of the study was to assess 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 4332 cells/mL. A water accommodated fraction (WAF)* with a nominal loading of 100 mg/L was tested. Six replicates were tested for the limit loading and the control. Environmental conditions were determined to be within the acceptable limits.

The test item Fatty acids, C16-18-, reaction products with diethanolaminewas analytically verified by LC-MS/MS at the start of the exposure (0 h) and at the end of the exposure (72 h) of the WAF with a nominal loading of 100 mg/L and the control. Details for the analytical method are presented in part 14.

The measured concentration of the test item in the WAF at the start of the exposure (0 h) was 0.660 mg/L. The measured concentration in the WAF at the end of the exposure (72 h) was 0.370 mg/L. All effect values are given based on the nominal loading of the test item.

NOEL, LOEL, EL-values of Fatty acids, C16-18-, reaction products with diethanolamine (0-72 hours)

                   based on the nominal loading with 100 mg test item/L

                               

	Growth Rate Inhibition
NOEL	100
LOEL	> 100
ErL10	> 100
ErL20	> 100
ErL50	> 100
	Yield Inhibition
NOEL	100
LOEL	100
EyL10	100
EyL20	100
EyL50	> 100

Description of key information

In this study caused slightly inhibition on the growth rate and yield of the freshwater green alga Desmodesmus subspicatus was found after 72 hours exposure to the test item Fatty acids, C16-18-, reaction products with diethanolamine. All effect values given are based on the nominal loading level of the test item Fatty acids, C16-18-, reaction products with diethanolamine.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

100 mg/L

EC10 or NOEC for marine water algae:

100 mg/L

Additional information

The toxicity ofFatty acids,C16-18-, reaction products with diethanolamine to the unicellular freshwater green algaDesmodesmus subspicatuswas determined according to the principles of OECD 201 (2006)atDr.U.Noack-Laboratorienin 31157 Sarstedt, Germanyfrom2013-03-11 to 2013-03-15, with the definitive exposure phase from2013-03-12 to 2013-03-15.The aim of the study was to assess 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 4332 cells/mL.A water accommodated fraction (WAF)*with a nominal loading of 100 mg/Lwas tested.Sixreplicates were tested for the limit loading and the control.Environmental conditionswere determined to be within the acceptable limits.

The test itemFattyacids, C16-18-, reactionproducts with diethanolaminewas analytically verified by LC-MS/MSat the start of the exposure(0 h) and at the end of the exposure (72 h) of the WAF with a nominal loading of 100 mg/L and the control. Details for the analytical method are presented inpart14.

The measured concentration of the test item in the WAF at the start of the exposure (0 h) was 0.660 mg/L. The measured concentration in the WAF at the end of the exposure (72 h) was 0.370 mg/L. The analytical results are presented inTable 8.All effect values are given based on the nominal loading of the test item.

 

Table1:  NOEL, LOEL, EL-values ofFatty acids, C16-18-, reaction products with diethanolamine(0-72 hours)

                  based on the nominal loading with 100 mg test item/L

                               

	Growth Rate Inhibition
NOEL	100
LOEL	> 100
ErL10	> 100
ErL20	> 100
ErL50	> 100
	Yield Inhibition
NOEL	< 100
LOEL	100
EyL10	< 100
EyL20	< 100
EyL50	> 100