Reaction products resulting from the esterification of C18 unsaturated fatty acid with glycerol and glycerol oligomers

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:

2015-02-09 to 2015-02-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented guideline study under GLP without deviations.

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L.
- Sampling method: All loading levels and the control were analytically verified via analysis of total organic carbon (TOC, according to DIN EN 1484) measured as NPOC (Non Purgeable Organic Carbon), at the start and at the end of the test. Separate replicates for the test item analysis after 0 and 72 hours were prepared without algae and incubated under test conditions.
- Sample storage conditions before analysis: None

Vehicle:

no

Details on test solutions:

Water accommodated fraction
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. At the completion of mixing and following a 1-hour settlement period, the test item phase was separated by siphoning 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).
Exposures are expressed in terms of the original concentration of the test item in water at the start of the mixing period (loading level) irrespective of the actual concentration of the test item in the WAF.

Preparation of the water accommodated fractions
Five water accommodated fractions (WAF) were prepared with nominal loadings of the test item in the range of 1.00 to 100 mg/L set up in a geometric series with a factor of 3.16: 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L.
For each loading level an appropriate amount of the test item was weighed. The test item was applied onto a glass slide. The glass slide with the test item was inserted into a brown glass flask with an appropriate amount of dilution water. These dispersions were shaken for 24 hours with 20 rpm at room temperature. After a separation phase of 1 hour, the aqueous phase or WAFs were removed by siphoning (from the approximate centre of the glass flask). The resulting water accommodated fractions (WAF) were used in the test.

Test loadings
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 loadings are based on the results of a non-GLP preliminary range finding test. For the preliminary test a stock solution was used with a test item loading level of 25 mg/L (24 hours magnetic stirring at 1100 rpm). Further test loading levels were diluted out of this stock solution.

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

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Test organism
Desmodesmus subspicatus CHODAT SAG 86.81 (formerly Scenedesmus subspicatus CHODAT)

Reason for the selection of the test system
According to the guideline, Desmodesmus subspicatus CHODAT SAG 86.81 is suitable for this type of study.

Origin
Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen

Cultivation at test facility
Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 35 - 70 µE•m-2•s-1 for 24 h per day.

Culture medium
Nutrient medium Z according to LÜTTGE et al. (1994)


ACCLIMATION
- Acclimation period: 4 days
- Culturing media and conditions (same as test or not): yes
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Hardness:

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

Test temperature:

min.: 20.0
max.: 24.0
mean: 22.0

pH:

Nominal Test Item Loading pH-value
[mg/L] Start; 0 hours End; 72 hours
100 7.66 7.50
31.6 7.68 7.71
10.0 7.71 7.98
3.16 7.79 8.04
1.00 7.91 8.09
Control 8.11 8.31

Dissolved oxygen:

not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

nominal: 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L

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 4998 cells/mL
- Control end cells density: Mean 470043 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: 71.3



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal. No self-fluorescence was found in the definitive test at the loading level 100 mg/L.



The results of the preliminary range finding test are presented below:

Results of the Range Finding Test (0-72 hours)
Nominal Test Item Loading
[mg/L] Growth Rate Inhibition
[%] Yield Inhibition
[%]
25.0 13 48
2.50 - 3 - 18
0.250 - 12 - 80
negative inhibition = increase of growth
TOC Content of the Test Media at the Start and the End of the Exposure in the Preliminary Test (non-GLP)

Nominal Test Item Loading
[mg/L] TOC mg/L as NPOC
Test start (without algae), 0 hours Test end (without algae), 72 hours
25.0 16.5 12.3
2.50 3.10 2.58
0.250 < 2.00 < 2.00
Control < 2.00 < 2.00

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

other: NOEL

Effect conc.:

31.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

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

Duration:

72 h

Dose descriptor:

other: EL10

Effect conc.:

47.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 39.1 - 63.8

Duration:

72 h

Dose descriptor:

other: EL20

Effect conc.:

60.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 46.7 - 81.2

Duration:

72 h

Dose descriptor:

other: EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

other: NOEL

Effect conc.:

31.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

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

Duration:

72 h

Dose descriptor:

other: EL10

Effect conc.:

42.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 31.6 - 94.8

Duration:

72 h

Dose descriptor:

other: EL20

Effect conc.:

48.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 33.1 - 95.7

Duration:

72 h

Dose descriptor:

other: EL50

Effect conc.:

69.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 35.5 - 98.5

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Other: The test solutions were concentration related turbid
- Any stimulation of growth found in any treatment: 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):

The toxicity of potassium dichromate (SIGMA, batch number MKBH9127V, purity 99.9 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Desmodesmus subspicatus was determined over a period of 72 hours from 2014-10-14 to 2014-10-17. 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 0.586 0.678 ± 0.414
95 % confidence interval 0.522 - 0.818
Yield Inhibition
EyC50 0.401 0.319 ± 0.141
95 % confidence interval 0.285 - 0.469
SD = Standard deviation

Reported statistics and error estimates:

EL-values and statistical analyses
EL10-, EL20- and EL50-values of the growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response. Calculation of the confidence intervals EL10-, EL20- and EL50-values were carried out using standard procedures.

NOEL, LOEL and statistical analyses
The values for NOEL/LOEL were determined by calculation of statistical significance of inhibition of growth rate and yield. As standard methods, 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.

Cell Densities

 

Nominal Test Item Loading	Replicate		Cell Density [cells/mL]
[mg/L]	No.		0 hours	24 hours		48 hours		72 hours
100	1		4998	17646		117093		107343
	2		4998	19756		87150		126436
	3		4998	16529		103943		150615
	Mean		4998	17977		102729		128131
31.6	1		4998	16745		112277		506066
	2		4998	20934		75125		410817
	3		4998	22563		121949		570562
	Mean		4998	20081		103117		495815
10.0	1		4998	24815		114460		635665
	2		4998	16005		108560		476894
	3		4998	25374		139487		600710
	Mean		4998	22065		120836		571090
3.16	1		4998	14874		96952		615610
	2		4998	16745		102895		552976
	3		4998	21744		110981		520577
	Mean		4998	17788		103609		563054
1.00	1		4998	16416		99360		610542
	2		4998	18456		105987		537946
	3		4998	26452		128689		579139
	Mean		4998	20441		111345		575876
Control		1	4998		19933		106529		480510
		2	4998		18040		74922		431521
		3	4998		14229		74207		409907
		4	4998		20180		83827		464441
		5	4998		17794		90602		483542
		6	4998		21008		127277		550334
		Mean	4998		18531		92894		470043

 

 

 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 [mg/L]	Replicate	Growth Rate		Growth Rate Inhibition	Yield		Inhibition of Yield
	No.	[d^-1]		[%]	[cells/mL]		[%]
100	1		1.02	32		102345	78
	2		1.08	29		121438	74
	3		1.14	25		145617	69
	Mean	(+)	1.08	29	(+)	123133	74
31.6	1		1.54	-2		501068	-8
	2		1.47	3		405819	13
	3		1.58	-4		565564	-22
	Mean	(-)	1.53	-1	(-)	490817	-6
10.0	1		1.62	-7		630667	-36
	2		1.52	0		471896	-1
	3		1.60	-6		595712	-28
	Mean	(-)	1.58	-4	(-)	566092	-22
3.16	1		1.60	-6		610612	-31
	2		1.57	-4		547978	-18
	3		1.55	-2		515579	-11
	Mean	(-)	1.57	-4	(-)	558056	-20
1.00	1		1.60	-6		605544	-30
	2		1.56	-3		532948	-15
	3		1.58	-5		574141	-23
	Mean	(-)	1.58	-5	(-)	570878	-23
Control	1		1.52			475512
	2		1.49			426523
	3		1.47			404909
	4		1.51			459443
	5		1.52			478544
	6		1.57			545336
	Mean		1.51			465045

negative inhibitions = increase of growth

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	1.38	1.68	1.51	1.52	0.147	9.66	15.0
	2	1.28	1.42	1.75	1.49	0.240	16.1
	3	1.05	1.65	1.71	1.47	0.367	25.0
	4	1.40	1.42	1.71	1.51	0.175	11.6
	5	1.27	1.63	1.67	1.52	0.221	14.5
	6	1.44	1.80	1.46	1.57	0.203	13.0
				Mean	1.51
				SD ±	0.03
				CV [%]	2.25

CV= Coefficient of variation

SD= Standard deviation

Validity criteria fulfilled:

yes

Conclusions:

In this study, Reaction mass of C18 (unsaturated) esters with glycerine oligomers was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 hours with the following effect values: The NOEL-values for both inhibition of growth rate and yield after 72 hours were 31.6 mg/L. Consequently, the LOEL-values for both parameters after 72 hours were 100 mg/L. The EL50-value for inhibition of growth rate (ErC50) after 72 hours was determined to exceed 100 mg/L. The corresponding EC20- and EC10-values for inhibition of growth rate (ErC20, ErC10) with 95 % confidence intervals after 72 hours were determined to be 60.9 (46.7 – 81.2) and 47.3 (39.1 – 63.8) mg/L, respectively.
The EC50-value for inhibition of yield (EyC50) with 95 % confidence intervals after 72 hours was 69.7 (35.5 – 98.5) mg/L. The corresponding EC20- and EC10-values for inhibition of yield (EyC20, EyC10) with 95 % confidence intervals after 72 hours were determined to be 48.4 (33.1 – 95.7) and 42.2 (31.6 – 94.8) mg/L, respectively.
All effect levels are given based on nominal test item loading levels of Reaction mass of C18 (unsaturated) esters with glycerine oligomers.

Executive summary:

The toxicity of Reactionmass of C18 (unsaturated) esters with glycerine oligomers (batch number: ESD0017954) to the unicellular freshwater green alga Desmodesmus subspicatuswas determined according to the principles of OECD 201 (2011) and Council Regulation (EC) No. 440/2008/ Method C.3 at Dr.U.Noack-Laboratorienin 31157 Sarstedt, Germany, from 2015-02-09 to 2015-02-13 with the definitive exposure phase from 2015-02-10 to 2015-02-13. The aim of the study was to assess the effects of the test item on growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 4998 cells/mL. Based on a preliminary test, 5 water accommodated fractions (WAF) with nominal loadings of the test item with a dilution factor of 3.16 were tested: 1.00 – 3.16 – 10.0 – 31.6 – 100 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

 

All test loading levels of Reaction mass of C18 (unsaturated) esters with glycerine oligomers and the control were analytically verified by analysis of total organic carbon (TOC), measured as NPOC (Non-Purgeable Organic Carbon) at the start (0 hours) and the end of exposure (72 hours).
All effect values given are based on the nominal test item loading levels of Reaction mass of C18 (unsaturated) esters with glycerine oligomers.

 

NOEL, LOEL, EL-values and Confidence Intervals ofReactionmass of C18 (unsaturated) esters with glycerine oligomers (0-72 hours)    

based on the nominal loadings of the test item [mg/L]

 

	Growth Rate Inhibition Nominal test item loading [mg/L]
NOEL	31.6
LOEL	100
ErL10	47.3 (39.1 – 63.8)
ErL20	60.9 (46.7 – 81.2)
ErL50	> 100
	Inhibition of Yield Nominal test item loading [mg/L]
NOEL	31.6
LOEL	100
EyL10	42.2 (31.6 – 94.8)
EyL20	48.4 (33.1 – 95.7)
EyL50	69.7 (35.5 – 98.5)

Description of key information

In the algae test (OECD201) no effects were observed up to the nominal loading rate of 31.6 mg/l corresponding to a TOC measured concentration on 14.6 mg/l. ErL50 has been determined to be >100mg/l nominal loading rate corresponding to a TOC measured concentration on 51.4 mg/l.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

31.6 mg/L