Alcohols, C6-8, 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:

2012-10-16 to 2012-10-23

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:

Alcohols, C6-C8-(even numbered, linear)-ethoxylated (<2,5 EO)

EC Number:

800-182-9

Cas Number:

1426148-68-6

Molecular formula:

R-O-R' , whereas R=C6-8-(even numbered, linear, saturated)-alkyl and R'=H or xEO wheras x=1-12, mean=2,3

IUPAC Name:

Alcohols, C6-C8-(even numbered, linear)-ethoxylated (<2,5 EO)

Details on test material:

- Name of test material (as cited in study report): Alcohols, C6-8 alkyl-(even, linear), ethoxylated (< 2.5 EO)
- Physical state: Liquid, colourless
- Analytical purity: 90 % (w/w)
- Purity test date: 2012-06-15
- Lot/batch No.: ESD0006420 (19.05.2011)
- Expiration date of the lot/batch: 2013-09-01
- 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: 1.00 - 3.20 - 10.0 - 32.0 - 100 mg/L
- Sampling method: The concentrations of the test item were analytically verified from all test concentrations and the control after 0 hours (test start) and 72 hours (test end). Samples at test start were taken from an additional replicate without algae. At end of the test samples were taken directly from the pooled test replicates.
- Sample storage conditions before analysis:All samples were stored at room temperature before preparation and before analysis, if necessary.

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: 1.00 - 3.20 - 10.0 - 32.0 - 100 mg/L
- Controls: Six replicates without test item were tested under the samte 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 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: 22 °C, Max: 23 °C

pH:

Nominal
test item concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
100 7.73 7.96
32.0 7.70 8.07
10.0 7.73 8.13
3.20 7.75 8.27
1.00 7.75 8.30
Control 7.95 8.28

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 788108 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 [%]
100 100 100
10.0 3 13
1.00 1 8

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

44.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI (41.3-50.2)

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

30.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI (29.7-32.0)

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: 0.540 (CI 0.528 - 0.551) mg/L
Yield Inhibition: 0.324 (CI 0.304 - 0.347) mg/L

Reported statistics and error estimates:

EC10-, EC20- and EC50-values of the growth rate inhibition were calculated by sigmoidal dose-response regression and the EC50 of the yield inhibition after 72 hours by linear dose-response regression. The calculation of the confidence intervals of the EC10-, EC20- and EC50-values for the growth rate inhibition and of the EC50 of the yield inhibition were carried from the best fit values, the standard error and the t-distribution with the software GraphPad Prism. The EC10- and EC20- values for the inhibition of yield are defined as the concentrations below and above the respective EC-value.
NOEC/LOEC was 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 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 concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 hours		24 hours	48 hours	72 hours
100		1		4332		11046	6288	5102
		2		4332		12958	7077	3193
		3		4332		12776	5976	2888
		Mean		4332		12260	6447	3728
32.0		1		4332		19833	70911	383990
		2		4332		9175^1)	13103^1)	12128^1)
		3		4332		18393	91291	445984
		Mean		4332		19113	81101	414987
10.0		1		4332		24994	192208	977300
		2		4332		18493	171425	854960
		3		4332		9964^1)	9669^1)	14518^1)
		Mean		4332		21744	181817	916130
3.20		1		4332		23783	169488	850620
		2		4332		20783	182659	897857
		3		4332		24906	174684	864866
		Mean		4332		23157	175610	871114
1.00		1		4332		25422	177225	830523
		2		4332		21755	181829	809357
		3		4332		20953	158594	810678
		Mean		4332		22710	172549	816853
Control		1		4332		21692	168670	787185
		2		4332		21450	190660	807030
		3		4332		23560	190132	730795
		4		4332		22692	161751	816496
		5		4332		28680	179967	811213
		6		4332		25139	178999	775926
		Mean		4332		23869	178363	788108

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		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
100	1		0.055	96.9		770	99.9
	2		-0.102	100		-1139	100
	3		-0.135	100		-1444	100
	Mean	(+)	-0.061	99.0	(+)	-604	100
32.0	1		1.495	13.8		379658	51.6
	2		n.a.	n.a.		n.a.	n.a.
	3		1.545	10.9		441652	43.7
	Mean	(+)	1.520	12.4	(+)	410655	47.7
10.0	1		1.806	-4.15		972968	-24.1
	2		1.762	-1.58		850628	-8.53
	3		n.a.	n.a.		n.a.	n.a.
	Mean	(+)^#	1.784	-2.87	(+)^#	911798	-16.3
3.20	1		1.760	-1.48		846288	-7.98
	2		1.778	-2.52		893525	-14.0
	3		1.766	-1.80		860534	-9.79
	Mean	(-)	1.768	-1.93	(+)^#	866782	-10.6
1.00	1		1.752	-1.02		826191	-5.41
	2		1.743	-0.52		805025	-2.71
	3		1.744	-0.56		806346	-2.88
	Mean	(-)	1.746	-0.70	(-)	812521	-3.67
Control	1		1.734			782853
	2		1.742			802698
	3		1.709			726463
	4		1.746			812164
	5		1.744			806881
	6		1.729			771594
	Mean		1.734			783776

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	1.61	2.05	1.54	1.734	0.277	16.0	16.2
	2	1.60	2.19	1.44	1.742	0.391	22.4
	3	1.69	2.09	1.35	1.709	0.371	21.7
	4	1.66	1.96	1.62	1.746	0.190	10.8
	5	1.89	1.84	1.51	1.744	0.208	11.9
	6	1.76	1.96	1.47	1.729	0.249	14.4
				Mean	1.73
				SD ±	0.01
				CV [%]	0.80

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study Alcohols, C6-8 alkyl-(even, linear), ethoxylated (<2.5 EO) was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 hours with the following effect values (nominal test item concentrations): the NOEC-values for both inhibition of growth rate and yield after 72 hours were 10.0 mg/L. The LOEC-values for both inhibition of growth rate and yield after 72 hours were 32.0 mg/L. The EC50-values for inhibition of specific growth rate (ErC50) and yield (EyC50) with 95 % confidence intervals after 72 hours were 44.6 (41.3 – 50.2) and 36.1 (32.0 – 40.7) mg/L, respectively.
All effect levels are given based on nominal concentrations of Alcohols, C6-8 alkyl-(even, linear), ethoxylated (<2.5 EO).

Executive summary:

The toxicity of Alcohols, C6-8 alkyl-(even, linear), ethoxylated (2.5 EO) (batch number: ESD0006420)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 2012-10-16 to 2012-10-23, with the definitive exposure phase from 2012-10-16 to 2012-10-19. 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 concentration levels were tested in a geometrical series with a dilution factor of √10 (nominal): 1.00 - 3.20 - 10.0 - 32.0 - 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.

The concentrations of Alcohols, C6-8 alkyl-(even, linear), ethoxylated (2.5 EO) were analytically verified by LC-MS/MS at test start and test end of all test concentrations and the control.

The measured concentrations of Alcohols, C6-8 alkyl-(even, linear), ethoxylated (2.5 EO) at test start were in the range of 100 to 110 % and after 72 hours in the range of 94 to 114 % of the nominal values. Therefore, all effect values given are based on the nominal concentrations of the test item Alcohols, C6-8 alkyl-(even, linear), ethoxylated (2.5 EO).

NOEC, LOEC, EC - values and 95 % Confidence Intervals of Alcohols, C6-8 alkyl-(even, linear), ethoxylated (2.5 EO) (0-72 hours)

                   based on nominal test item concentrations [mg/L]

	Growth Rate Inhibition
NOEC	10.0
LOEC	32.0
ErC10	30.8 (29.7 – 32.0)
ErC20	35.1 (34.0 – 37.1)
ErC50	44.6 (41.3 – 50.2)
	Yield Inhibition
NOEC	10.0
LOEC	32.0
EyC10	10.0 – 32.0
EyC20	10.0 – 32.0
EyC50	36.1 (32.0 – 40.7)