C18 (branched) fatty acids, esters with mono-, di-, and tri-glycerol

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:

2013-01-11 to 2013-01-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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:

C18 (branched) fatty acids, esters with mono-, di-, and tri-glycerol

EC Number:

701-479-5

Molecular formula:

C42H82O7

IUPAC Name:

C18 (branched) fatty acids, esters with mono-, di-, and tri-glycerol

Details on test material:

- Name of test material (as cited in study report): Diglycerol diisostearate
- Physical state: Viscous liquid, light yellow
- Analytical purity: 100 % (w/w) UVCB
- Purity test date: 2012-02-29
- Lot/batch No.: ESD0012304
- Expiration date of the lot/batch: 2013-06-09
- Stability under test conditions: Stable
- Storage condition of test material: Keep container tightly closed and dry.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Five water accommodated fractions (WAF): 6.40 - 16.0 - 40.0 - 100 - 250 mg/L
- Sampling method: All test item loadings and the control were analytically verified via analysis of organic carbon (TOC, according to DIN EN 1484) 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. Results below 2 mg/L were given as < 2 mg/L.
- Sample storage conditions before analysis: None, direct analysis

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Five water accommodated fractions (WAF) were prepared with nominal loadings of the test item in the range of 6.40 to 250 mg/L set up in a geometric series with a factor of 2.5:
6.40 - 16.0 - 40.0 - 100 - 250 mg/L.
For each loading level, an appropriate amount of the test item was weighed out. The test item was applied onto a glass slide. The glass slide with the test item was inserted in a brown glass flask with an appropriate amount of dilution water. These dispersions were stirred for at least 72 hours with 1100 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 loading rate WAF.
- Eluate: DIlution water
- Differential loading: 6.40 - 16.0 - 40.0 - 100 - 250 mg/L
- Controls: Six replicates (without test item) were tested under the same test conditions as the test vessels.

Test organisms

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): 3 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.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Test conditions

Hardness:

0.24 mmol Ca+Mg/l

Test temperature:

min.: 21.5 max.: 22.5, mean value: 22.0

pH:

Nominal test item loading pH-value
[mg/L] Start; 0 hours End; 72 hours
250 7.58 7.92
100 7.62 7.96
40.0 7.75 7.99
16.0 7.83 8.00
6.40 7.99 8.16
Control 8.10 8.21

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 4910 cells/mL
- Control end cells density: Mean 776146 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 first Study (0-72 hours)

Water
accommodated
fraction [mg/L] Growth Rate Inhibition [%] Yield Inhibition [%]
100 12 44
40.0 7 28
16.0 1 7
6.40 -3 -14
2.56 -3 -18

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

250 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 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. 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 a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.

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
250	1	4332	23013	157619	939592
	2	4332	18364	136654	1105931
	3	4332	18154	133698	936981
	Mean	4332	19844	142657	994168
100	1	4332	18588	115727	725323
	2	4332	13968	142076	894964
	3	4332	14072	133232	755452
	Mean	4332	15543	130345	791913
40.0	1	4332	16742	136862	642515
	2	4332	15691	123124	737117
	3	4332	16327	101418	619273
	Mean	4332	16253	120468	666302
16.0	1	4332	20207	87542	527251
	2	4332	14537	139567	603894
	3	4332	11615	111356	553165
	Mean	4332	15453	112822	561437
6.40	1	4332	17824	145863	1013719
	2	4332	23859	147265	1128984
	3	4332	19078	126974	999630
	Mean	4332	20254	140034	1047444
Control	1	4332	21047	138579	836970
	2	4332	18305	146360	815427
	3	4332	21201	127433	860479
	4	4332	15122	127622	663461
	5	4332	19591	128075	780109
	6	4332	13845	128741	700430
	Mean	4332	18185	132802	776146

Evaluation after 72 hours

                   Statistically significant differences of growth rates and yield compared to

                   control values are marked (+), non significant differences are marked (-).

Nominal test item loading	Replicate	Growth rate		Inhibition of growth rate	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
250	1		1.79	-3.77		935260	-21.2
	2		1.85	-6.92		1101599	-42.7
	3		1.79	-3.72		932649	-20.8
	Mean	(+)^#	1.81	-4.80	(+)^#	989836	-28.3
100	1		1.71	1.22		720991	6.58
	2		1.78	-2.83		890632	-15.4
	3		1.72	0.44		751120	2.68
	Mean	(-)	1.74	-0.39	(-)	787581	-2.04
40.0	1		1.67	3.56		638183	17.3
	2		1.71	0.91		732785	5.06
	3		1.65	4.27		614941	20.3
	Mean	(-)	1.68	2.91	(-)	661970	14.2
16.0	1		1.60	7.37		522919	32.3
	2		1.65	4.76		599562	22.3
	3		1.62	6.45		548833	28.9
	Mean	(+)	1.62	6.19	(+)	557105	27.8
6.40	1		1.82	-5.24		1009387	-30.8
	2		1.85	-7.31		1124652	-45.7
	3		1.81	-4.97		995298	-29.0
	Mean	(+^#)	1.83	-5.84	(+)^#	1043112	-35.2
Control	1		1.76			832638
	2		1.75			811095
	3		1.76			856147
	4		1.68			659129
	5		1.73			775777
	6		1.70			696098
	Mean		1.73			771814

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	1.58	1.89	1.80	1.76	0.157	8.93	17.6
	2	1.44	2.08	1.72	1.75	0.320	18.3
	3	1.59	1.79	1.91	1.76	0.163	9.24
	4	1.25	2.13	1.65	1.68	0.442	26.4
	5	1.51	1.88	1.81	1.73	0.196	11.3
	6	1.16	2.23	1.69	1.70	0.534	31.5
				Mean	1.73
				SD ±	0.03
				CV [%]	2.00

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study no inhibition on growth of the freshwater green alga Desmodesmus subspicatus was found after 72 hours exposure to the test item Diglycerol diisostearate. The nominal EL50-values for inhibition of growth rate (ErL50) and yield (EyL50) after 72 hours were both > 250 mg/L, respectively. The nominal NOEL-values for inhibition of growth rate and yield after 72 hours were for both 250 mg/L, respectively.
All effect values given are based on nominal test item loadings of Diglycerol diisostearate.

Executive summary:

The toxicity of Diglycerol diisostearate (batch number: ESD0012304)to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorienin 31157 Sarstedt, Germany from 2013-01-11 to 2013-01-31 with the definitive exposure phase from 2013-01-28 to 2013-01-31. 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. Five water accommodated fraction (WAF) were prepared with a nominal loading of 6.40 - 16.0 - 40.0 - 100 - 250 mg test item/L and removal of particulate matter by sedimentation. Three replicates were used for each water accommodated fraction and six for the control. The environmental conditions were within the acceptable limits.

All test loadings of Diglycerol diisostearate, and the control were analytically verified via analysis of total organic carbon (TOC) at the start and the end of the exposure.

All effect values given are based on nominal test item loadings of Diglycerol diisostearate. The growth inhibition results of the test item are summarized in Table 1.

NOEL, LOEL, EL - values and 95 % Confidence Intervals of Diglycerol diisostearate
(0-72 hours)

                   based on nominal test item loadings [mg/L]

	Inhibition of Growth Rate
NOEL	250
LOEL	>250
ErL10	>250
ErL20	>250
ErL50	>250
	Inhibition of Yield
NOEL	250
LOEL	>250
EyL10	>250
EyL20	>250
EyL50	>250