Fatty acids, C16-18 (even numbered), esters with 1,2,3-propanetriol and oligomers, ethoxylated

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:

from 2017-03-06 to 2017-03-10 at the test facility with the definitive exposure phase from 2017-03-07 to 2017-03-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C16-18 (even numbered), esters with 1,2,3-propanetriol and oligomers, ethoxylated

IUPAC Name:

Fatty acids, C16-18 (even numbered), esters with 1,2,3-propanetriol and oligomers, ethoxylated

Test material form:

solid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Type of analysis and analysis dates
The limit concentration and the control were analytically verified via analysis of total organic carbon (TOC, according to DIN EN 1484) at the start and at the end of the exposure. Separate replicates for the test item analysis after 0 and 72 hours were prepared without algae and incubated under test conditions.

Test solutions

Vehicle:

no

Details on test solutions:

Water Accommodated Fraction (WAF)
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000) is to expose the organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted solvents. Using this approach, aqueous media were prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase. After completion of mixing and following a settlement period, the test item phase was separated by siphon and the test organisms were exposed to the aqueous phase, the WAF (which may contain dissolved and/or suspended and/or emulsified fractions of the test item mixture). Exposure was expressed in terms of the original concentration of the test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of the test item in the WAF.

Preparation of test solutions
For the single water accommodated fraction (WAF) 100 mg/L of the test item were weighed out and transferred into a brown glass flask with dilution water one day prior to application. The WAF was shaken with 20 rpm for 24 hours at room temperature. After a separation phase of 1 hour the WAF was taken from the homogeneous liquid phase from the middle of the water body. The WAF was checked after stirring via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). The Tyndall effect was positive.

Test loading
Per definition of the WAF, all terms related to concentration level are given as loading level because partly dissolved compounds and mixtures cannot be related to concentrations.
The limit loading is based on the results of a non-GLP preliminary ran DGSB DGSge finding test.

Test concentrations
One limit loading of 100 mg/L was tested. The limit loading is 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

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 Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 2567 – 5130 lux corresponding to 35 - 70 µE ∙ m-2 ∙ s-1 for 24 h per day.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No

Test conditions

Hardness:

Not specified

Test temperature:

min.: 22.0 max.: 22.5 mean value: 22.3

pH:

Nominal test item loading pH-value
[mg/L] Start; 0 hours End; 72 hours
100 8.09 9.24
Control 8.05 9.32

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 6839 cells/mL
- Control end cells density: Mean 1949178 cells/mL
- No. of vessels per concentration (replicates): 6
- 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 0.064
Na2EDTA 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: 5631 lux,



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
-Chlorophyll a-fluorescence
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 preliminary range finding test.

Reference substance (positive control):

yes

Remarks:

(Potassium dichromate)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

other: NOEL

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

other: LOEL

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Duration:

72 h

Dose descriptor:

other: NOEL

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 7

Duration:

72 h

Dose descriptor:

other: LOEL

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 8

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 9

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 10

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

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 item concentrations were clear during the exposure. The algae in the saturated solution were agglutinated after 72 hours. No agglutination of the algae was observed in the control replicates.
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

The toxicity of potassium dichromate (SIGMA, 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 2016-10-11 to 2016-10-14 (with headspace). 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 0.435 0.764 ± 0.513
95 % confidence interval 0.414 – 0.458
Yield inhibition
EyC50 0.230 0.402 ± 0.293
95 % confidence interval 0.179 – 0.322
SD = Standard deviation

Reported statistics and error estimates:

EL-values and statistica analyses
EL-values of the growth rate and yield inhibition were estimated empirically based on the results of the only treatment level (Limit test design).
 
NOEL and LOEL values
NOEL/LOEL were determined by calculation of statistical significance of growth rate and yield. As a standard, a t-test was used for NOEL/LOEL calculations. When running a t-test a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normal distribution of data. 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.

Software
The data for the tables in this report are 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.

Any other information on results incl. tables

Cell Densities

Nominal test item loading		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 hours		24 hours		48 hours		72 hours
100		1		6839		29580		312536		1792940
		2		6839		32117		366351		1461190
		3		6839		29761		282492		1796217
		4		6839		30981		328395		2092913
		5		6839		34253		342732		1856785
		6		6839		29793		338858		1604681
		Mean		6839		31081		328561		1767454
Control		1		6839		29513		297777		1968324
		2		6839		31590		400959		2159041
		3		6839		33153		410884		2206032
		4		6839		23939		250692		1624695
		5		6839		34364		379939		1858365
		6		6839		25852		307608		1878613
		Mean		6839		29735		341310		1949178

 

 

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		1.86	1		1786101	8
	2		1.79	5		1454351	25
	3		1.86	1		1789378	8
	4		1.91	-1		2086074	-7
	5		1.87	1		1849946	5
	6		1.82	3		1597842	18
	Mean	(-)	1.85	2	(-)	1760615	9
Control	1		1.89			1961485
	2		1.92			2152202
	3		1.93			2199193
	4		1.82			1617856
	5		1.87			1851526
	6		1.87			1871774
	Mean		1.88			1942339

 

 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	1.46	2.31	1.89	1.89	0.425	22.5	27.2
	2	1.53	2.54	1.68	1.92	0.545	28.4
	3	1.58	2.52	1.68	1.93	0.515	26.7
	4	1.25	2.35	1.87	1.82	0.549	30.1
	5	1.61	2.40	1.59	1.87	0.463	24.8
	6	1.33	2.48	1.81	1.87	0.576	30.8
				Mean	1.88
				SD ±	0.04
				CV [%]	1.98

SD = Standard deviation CV = Coefficient of variation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, no inhibition of growth on the freshwater green alga Pseudokirchneriella subcapitata was found after 72 hours exposure to the test item . The nominal EL50-values for inhibition of growth rate (ErL50) and yield (EyL50) after 72 hours were >100 mg/L, respectively. The nominal NOEL-values for inhibition of growth rate and yield after 72 hours were 100 mg/L, respectively. All effect values given are based on nominal test item loadings.

Executive summary:

The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009/Method C.3 from 2017-03-06 to 2017-03-10 at the test facility with the definitive exposure phase from 2017-03-07 to 2017-03-10. 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 6839 cells/mL. A water accommodated fraction with a nominal loading of 100 mg test item/L was tested. Six replicates were used for the limit loading level and the control. The environmental conditions were within the acceptable limits.

 

The concentrations of the test item were analytically verified via analysis of total organic carbon (TOC, according to DIN EN 1484) at the start of the exposure (fresh media, 0 hours) and at the end of the exposure (old media, 72 hours). All effect values were based on the nominal test item loading of the test item. The results are summarized in the following table:

 

NOEL, LOEL, EL - values (0-72hours)

based on the nominal test loading [mg/L]

 

	Inhibition of Growth Rate                                                nominal test item loading [mg/L]
NOEL	100
LOEL	>100
ErL10	>100
ErL20	>100
ErL50	>100
	Inhibition of Yield                                                          nominal test item loading [mg/L]
NOEL	100
LOEL	>100
EyL10	>100
EyL20	>100
EyL50	>100