Octanoic acid, esters with anhydrosorbitol and dianhydrosorbitol

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:

2012-11-20 to 2012-11-23

Reliability:

1 (reliable without restriction)

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:

Sorbitanmonocaprylate

IUPAC Name:

Sorbitanmonocaprylate

Details on test material:

- Name of test material (as cited in study report): Sorbitan monocaprylate
- Physical state: Viscous liquid, brown, clear
- Analytical purity: 99.7 % (w/w)
- Purity test date: 2011-09-21
- Lot/batch No.: DEG4149186
- Expiration date of the lot/batch: 2013-07-06
- Stability under test conditions: Stable in water and light at room temperature
- Storage condition of test material: Room temperature, protected from light, in the original container

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L
- Sampling method: All test item concentrations 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.
- 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: A stock solution of 100 mg/L was freshly prepared with dilution water and agitated.
- Eluate: Dilution water
- Differential loading: 6.25 - 12.5 - 25.0 - 50.0 - 100 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 SAG 86.81
- 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 from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 h 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: 21.5 °C, Max: 23.5 °C

pH:

Nominal test item concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
100 7.49 8.04
50.0 7.55 8.06
25.0 7.60 8.14
12.5 7.66 8.22
6.25 7.76 8.32
Control 7.98 8.40

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods"

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): sealed with 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: 4332 cells/mL
- Control end cells density: Mean 714201 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

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

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



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 hours)

Nominal Test Item Concentration
[mg/L] Growth Rate Inhibition [%] Yield Inhibition [%]
100 72 98
10 1 5
1 -7 -43

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

79.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI (74.7-84.5 mg/L)

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

16.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI (13.1 - 19.3 mg/L)

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: The stock solution was slightly turbid. After 24 hours a concentration-related agglutination of the test item was observed in the two highest concentration levels.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.631 (CI 0.583 - 0.687) mg/L
Yield Inhibition: 0.256 (CI 0.247 - 0.265) mg/L

Reported statistics and error estimates:

EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression. The calculation of the confidence intervals for the EC10-, EC20- and EC50-values were carried out from the best fit values, the standard error and the t-distribution.
NOEC/LOEC 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 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 a=0.05.
The Normality Test failed for yield calculation. Transformation (y = Log (y)) was carried out to pass the Normality Test.

Any other information on results incl. tables

Cell Densities

Nominal test item concentration	Replicate	Cell density [cells/mL]
[mg/L]	No.	0 hours	24 hours	48 hours	72 hours
100	1	4332	9722	21588	27592
	2	4332	12713	24390	33121
	3	4332	8838	24585	32419
	Mean	4332	10424	23521	31044
50.0	1	4332	8983	38257	156411
	2	4332	9247	31360	132390
	3	4332	10040	33379	144479
	Mean	4332	9423	34332	144427
25.0	1	4332	11839	66577	338255
	2	4332	12911	74377	296930
	3	4332	12087	71182	291866
	Mean	4332	12279	70712	309017
12.5	1	4332	13185	80422	426944
	2	4332	12961	72874	428705
	3	4332	12785	66118	433769
	Mean	4332	12977	73138	429806
6.25	1	4332	13367	106110	680179
	2	4332	13260	131037	730688
	3	4332	15298	129880	706409
	Mean	4332	13975	122342	705759
Control	1	4332	18107	146920	877434
	2	4332	15509	122741	640584
	3	4332	16518	136214	750565
	4	4332	17437	133069	724209
	5	4332	19396	124741	652189
	6	4332	13851	104396	640222
	Mean	4332	16803	128014	714201

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 concentration	Replicate	Growth rate		Inhibition of growth rate	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
100	1		0.617	63.7		23260	96.7
	2		0.678	60.1		28789	95.9
	3		0.671	60.5		28087	96.0
	Mean	(+)	0.655	61.4	(+)	26712	96.2
50.0	1		1.20	29.7		152079	78.6
	2		1.14	32.9		128058	82.0
	3		1.17	31.2		140147	80.3
	Mean	(+)	1.17	31.3	(+)	140095	80.3
25.0	1		1.45	14.5		333923	53.0
	2		1.41	17.1		292598	58.8
	3		1.40	17.4		287534	59.5
	Mean	(+)	1.42	16.3	(+)	304685	57.1
12.5	1		1.53	9.96		422612	40.5
	2		1.53	9.88		424373	40.2
	3		1.54	9.65		429437	39.5
	Mean	(+)*	1.53	9.83	(+)	425474	40.1
6.25	1		1.69	0.83		675847	4.79
	2		1.71	-0.58		726356	-2.32
	3		1.70	0.09		702077	1.10
	Mean	(-)	1.70	0.11	(-)	701427	1.19
Control	1		1.77			873102
	2		1.67			636252
	3		1.72			746233
	4		1.71			719877
	5		1.67			647857
	6		1.67			635890
	Mean		1.70			709869

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	1.43	2.09	1.79	1.77	0.332	18.8	20.3
	2	1.28	2.07	1.65	1.67	0.397	23.8
	3	1.34	2.11	1.71	1.72	0.386	22.5
	4	1.39	2.03	1.69	1.71	0.320	18.8
	5	1.50	1.86	1.65	1.67	0.182	10.9
	6	1.16	2.02	1.81	1.67	0.448	26.9
				Mean	1.70
				SD ±	0.04
				CV [%]	2.43

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, Sorbitan monocaprylate, was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus over the period of 72 hours. The nominal EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 79.8 (74.7 – 84.5) and 17.5 (15.4 – 19.7) mg test item/L, respectively. The nominal NOEC-values for inhibition of growth rate and yield after 72 hours were 12.5 and 6.25 mg test item/L, respectively. For other endpoints, please refer to Table1.
All effect values given are based on nominal test item concentrations of Sorbitan monocaprylate.

Executive summary:

The toxicity of Sorbitan monocaprylate (batch number: DEG4149186)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 2012-11-20 to 2012-11-23. 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. Based on a preliminary test, 5 concentrations were tested in a geometrical series with a dilution factor of 2, nominal: 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. Environmental conditions were within the acceptable limits.

All test concentrations of Sorbitan monocaprylate, and the control were analytically verified via analysis of organic carbon (TOC) at the start and the end of the exposure.

All effect values given are based on nominal test item concentrations of Sorbitan monocaprylate. The growth inhibition results of the test item are summarized in Table 1.

Table 1:    NOEC, LOEC, EC - values and 95 % Confidence Intervals of Sorbitan monocaprylate
(0-72 hours)

                   based on nominal test item concentrations [mg/L]

	Inhibition of Growth Rate
NOEC	12.5
LOEC	25.0
ErC10	16.3 (13.1 – 19.3)
ErC20	31.5 (28.5 – 34.8)
ErC50	79.8 (74.7 – 84.5)
	Inhibition of Yield
NOEC	6.25
LOEC	12.5
EyC10	7.33 (6.46 – 8.13)
EyC20	9.00 (8.12 – 10.1)
EyC50	17.5 (15.4 – 19.7)