Reaction mass of 5,8,11,14-Tetraoxaoctadecane and 5,8,11,14,17-Pentaoxaheneicosane

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:

2012-06-04 to 2012-06-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study, no deficiencies

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

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:

5,8,11,14,17-pentaoxahenicosane; 5,8,11,14-tetraoxaoctadecane

EC Number:

940-417-3

Molecular formula:

C14H30O4 C16H34O5

IUPAC Name:

5,8,11,14,17-pentaoxahenicosane; 5,8,11,14-tetraoxaoctadecane

Details on test material:

- Name of test material (as cited in study report): Polyglycol BB 300
- Physical state: Liquid, colourless
- Analytical purity: 99.6 % (a/a)
- Purity test date: 2011-06-28
- Lot/batch No.: DEG4155254
- Expiration date of the lot/batch: 2013-06-24
- Stability under test conditions: Not applicable
- Storage condition of test material: Room temperature, protected from light, in original container.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 10.2 - 25.6 - 64.0 - 160 - 400 mg/L (factor 2.5)
- 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 additional replicates without algae. At the end of the test samples were taken directly from pooled test replicates.
- Sample storage conditions before analysis: All original samples were stored at 6 ± 2 °C until sample preparation, if necessary. Prepared samples were stored at room temperature until 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 400 mg/L, freshly prepared with dilution water was agitated.
- Eluate: Dilution water
- Differential loading: 10.2 - 25.6 - 64.0 - 160 - 400 mg/L (factor 2.5)
- Controls: Six replicates (without test item) were tested under the same test conditions as the test replicates.

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:

Dilution water: 0.24 mmol Ca+mg/L.

Test temperature:

min.: 21.0 max.: 22.5, mean value: 21.8

pH:

Nominal test item concentration pH-value
[mg/L] No. End; 72 hours
400 7.80 8.08
100 7.80 8.22
64.0 7.81 8.51
25.6 7.82 8.87
10.2 7.83 9.17
Control 7.92 9.27

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

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): 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: 907057 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: Dilution water, composition acc. to the guideline
- Hardness: 0.24 mmol Ca+Mg/L
- pH-value: 8.1 +/- 0.2
- Culture medium: Nutrient Medium Z acc. to LÜTTGE et al. (1994)


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: 69.2



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
- Range finding study:
Results of the Range Finding Test (0-72 hours)
Nominal Test Item Concentration
[mg/L] Growth Rate Inhibition [%] Yield Inhibition [%]
10000 100 100
2000 100 100
400 97 100
80 75 98
10 2 12
1.00 -2 -11
0.100 -1 -6

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

74.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI 70.9-78.8 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

29.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI 24.0-34.3 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: 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 0.540 0.686 ± 0.470
95 % confidence interval 0.528 - 0.551
Yield inhibition
EyC50 0.324 0.319 ± 0.140
95 % confidence interval 0.304 - 0.347
SD = Standard deviation

Reported statistics and error estimates:

NOEC/LOEC 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 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 =0.05.

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
400	1	4910	5851	4503	2665
	2	4910	4158	4016	2130
	3	4910	6121	3843	2696
	Mean	4910	5377	4121	2497
160	1	4910	9225	8972	8156
	2	4910	8288	6050	7871
	3	4910	8095	8027	6737
	Mean	4910	8536	7683	7588
64.0	1	4910	15186	42943	131278
	2	4910	12803	42652	123946
	3	4910	14171	46037	104794
	Mean	4910	14053	43877	120006
25.6	1	4910	22197	121414	558709
	2	4910	24224	132362	476081
	3	4910	24624	126160	474050
	Mean	4910	23682	126645	502947
10.2	1	4910	26458	173374	849345
	2	4910	25612	151947	744952
	3	4910	27660	176360	842677
	Mean	4910	26577	167227	812325
Control	1	4910	30764	170876	892707
	2	4910	25660	172826	930280
	3	4910	30564	189331	1021845
	4	4910	26804	174762	855540
	5	4910	25754	175175	992768
	6	4910	25240	198024	754599
	Mean	4910	27464	180166	907957

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 concentration	Replicate	Growth rate		Growth rate inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
400	1		0.00	100		0	100
	2		0.00	100		0	100
	3		0.00	100		0	100
	Mean	(+)	0.00	100	(+)	0	100
160	1		0.169	90.3		3246	99.6
	2		0.157	91.0		2961	99.7
	3		0.105	93.9		1827	99.8
	Mean	(+)	0.144	91.7	(+)	2678	99.7
64.0	1		1.10	37.0		126368	86.0
	2		1.08	38.1		119036	86.8
	3		1.02	41.3		99884	88.9
	Mean	(+)	1.06	38.8	(+)	115096	87.3
25.6	1		1.58	9.22		553799	38.7
	2		1.53	12.3		471171	47.8
	3		1.52	12.4		469140	48.1
	Mean	(+)	1.54	11.3	(+)	498037	44.9
10.2	1		1.72	1.18		844435	6.49
	2		1.67	3.70		740042	18.1
	3		1.72	1.34		837767	7.23
	Mean	(-)	1.70	2.07	(-)	807415	10.6
Control	1		1.73			887797
	2		1.75			925370
	3		1.78			1016935
	4		1.72			850630
	5		1.77			987858
	6		1.68			749689
	Mean		1.74			903047

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	1.84	1.72	1.65	1.73	0.0925	5.33	9.23
	2	1.65	1.91	1.68	1.75	0.139	7.94
	3	1.83	1.82	1.69	1.78	0.0810	4.55
	4	1.70	1.88	1.59	1.72	0.145	8.41
	5	1.66	1.92	1.74	1.77	0.133	7.54
	6	1.64	2.06	1.34	1.68	0.363	21.6
				Mean	1.74
				SD ±	0.04
				CV [%]	2.11

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study Polyglycol BB 300 was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 hours. The NOEC-value for inhibition of growth rate and inhibition of yield after 72 hours was 10.2 mg/L. The LOEC-value for inhibition of growth rate and inhibition of yield after 72 hours was 25.6 mg/L. The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) with 95 % confidence intervals after 72 hours were 74.6 (70.9 - 78.8) and 28.1 (25.9 - 30.3) mg/L, respectively.
All effect levels are given based on nominal concentrations of Polyglycol BB 300.

Executive summary:

The toxicity of PolyglycolBB 300 (batch number: DEG4155254) 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 2012-06-04 to 2012-06-12, with the definitive exposure phase from 2012-06-04 to 2012-06-07. 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 4910 cells/mL. Based on a preliminary test 5 concentration levels were tested in a geometrical series with a nominal dilution factor of 2.5: 10.2 - 25.6 - 64.0 - 160 - 400 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.

The concentrations of Polyglycol BB 300 were analytically verified by GC-MS at test start and test end of all test concentrations and the control.
The measured concentrations of PolyglycolBB 300 at test start were in the range of 99 to 114 % and after 72 hours in the range of 95 to 112 % of the nominal values. Therefore, all effect values given are based on the nominal test item concentrations of the test item Polyglycol BB 300.

NOEC, LOEC, EC - values and 95 % Confidence Intervals of Polyglycol BB 300 (0-72 hours)

                   based on nominal test item concentrations [mg/L]

	Growth Rate Inhibition
NOEC	10.2
LOEC	25.6
ErC10	29.3 (24.0 - 34.3)
ErC20	43.6 (39.6 - 47.5)
ErC50	74.6 (70.9 - 78.8)
	Inhibition of Yield
NOEC	10.2
LOEC	25.6
EyC10	9.83 ( 10.2 - 12.2)
EyC20	14.9 (13.0 - 17.0)
EyC50	28.1 (25.9 - 30.3)