Alcohols, C6-8, ethoxylated

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-08-22 to 2012-08-23

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 209 (Activated Sludge, Respiration 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:

no

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 5 g/L was prepared and agitated.
- Eluate: Synthetic waste water according to OECD Guidelinie 209
- Differential loadings: 10 - 32 - 100 - 320 - 1000 mg/L
- Controls: Test medium without test or reference item.

Test organisms

Test organisms (species):

activated sludge

Details on inoculum:

- Preparation of inoculum for exposure: Non adapted activated sludge from the sewage plant at Hildesheim (Municipal sewage treatment plant of 31137 Hildesheim, Germany)
- Pretreatment: Washed twice with chlorine free tap water. Adjustment to a dry sludge concentration of 3.0 g/L ± 10 % was not necessary. The sludge was used within 24 h after sampling.
- Dry sludge concentration: 2.94 g/L, corresponding to 1.47 g/L in the test vessel

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test conditions

Hardness:

Not determined

Test temperature:

19.6 °C

pH:

pH-value of the activated sludge 7.88
pH-value of the synthetic waste water 7.55

Salinity:

Not determined

Nominal and measured concentrations:

10 - 32 - 100 - 320 - 1000 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: 1000 mL Erlenmeyer flask
- Aeration: Shaking of flasks at 150 rpm to keep the dissolved oxygen concentration above 60 - 70 % saturation and to maintain the sludge flakes in suspension.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- Composition of test medium:
Test item concentration [mg/L]
10 32 100 320 1000
Inoculum [mL] 250
Synthetic waste water [mL] 16
Stock solution [mL] 1.0 3.2 10 32 100
Demineralised water [mL] 233 230.8 224 202 134

Test / reference item (direct weighing)
Reference item concentration [mg/L]
10 32 1000
Inoculum [mL] 250
Synthetic waste water [mL] 16
Stock solution [µL] 97 167 300
Demineralised water [mL] 233.9 233.83 233.7


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
After 3 h incubation, the oxygen concentration of each control, reference item and test item replicate was determined by means of oximeter immediately after transfering the solution to the measuring cell and the oxygen depletion was recorded for at least 3 minutes on a flatbed recorder.

TEST CONCENTRATIONS

- Range finding studies:

Inhibition of Respiration (Preliminary Tests)
Nominal Test Item Concentration pH pH Inhibition [%]
[mg/L] (test item in water) (test item in medium)
1 - - -1
10 - - 1
100 - - 2
10 - - 7
100 - - 6
1000 - - 63
1000 (6.29)
7.80* 7.77 65

Reference substance (positive control):

yes

Remarks:

Copper (II) sulphate pentahydrate p.a.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

618 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% CI (575-661 mg/L)

Effect concentrations 2

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: EC50: 114 mg/L

Reported statistics and error estimates:

The NOEC was determined by calculation of statistical significance of the inhibition of respiration in comparison to the control. One Way Analysis of Variance (ANOVA) and DUNNETT’s test was used for NOEC 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 test are 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.

The EC-values of the test item and the reference item were calculated by linear regression using software GraphPadPrism. Calculations of the confidence intervals for the EC-values were carried out using standard procedures.

Any other information on results incl. tables

From the oxygen depletion the oxygen uptake rates were calculated in mg O₂/L×h, additionally the specific respiration rates based on the dry sludge concentration were calculated in mg O₂/g×h.

The inhibition of the oxygen uptake rates of the test and reference item concentrations in % was calculated in comparison with the oxygen uptake rates of the control.

Oxygen Uptake Rates, Specific Respiration Rates and Inhibition of the Respiration
of the Control, Test and Reference Item Concentrations

	Test Item Concentration	Repl.	Oxygen Uptake Rate R [mg O2/L×h]	Specific Respiration Rate Rs [mg O2/g×h]	Inhibition [%]	Mean Inhibition [%]
	[mg/L]
	Control	1	26.8	18.2	–	–
		2	25.6	17.4
		3	28.8	19.6
		4	28.4	19.3
		5	29.2	19.9
		6	27.2	18.5
Test Item	10	1	28.0		-1	-3
		2	28.8		-4
		3	27.6		0
		4	29.6		-7
		5	28.0		-1
	32	1	28.8		-4	-7
		2	29.2		-5
		3	30.8		-11
		4	30.4		-10
		5	29.6		-7
	100	1	30.4		-10	-4
		2	27.6		0
		3	28.8		-4
		4	28.0		-1
		5	29.2		-5
	320	1	32.8		-18	-19
		2	31.6		-14
		3	33.6		-21
		4	34.8		-26
		5	32.0		-16
	1000	1	19.6		29	31
		2	19.2		31
		3	19.2		31
		4	18.4		34
		5	19.2		31
Reference Item	58	1	24.4		12	15
		2	23.6		15
		3	22.4		19
	100	4	16.0		42	42
		5	-^#		-
		2	16.0		42
	180	3	7.2		74	74
		4	7.2		74
		5	6.8		75

Coefficient of variation of oxygen uptake rates of the control: 5.0 %

Suspended solids in the test vessel: 1.47 g/L
^#) Replicate not measured, Erlenmeyer flask broke before measurement

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The EC10 of Alcohols, C6-8 alkyl-(even, linear), ethoxylated (<2.5 EO) is 618 mg/L. Alcohols, C6-8 alkyl-(even, linear), ethoxylated (<2.5 EO) is not toxic up to the concentration of 618 mg/L to activated sludge of a municipal sewage treatment plant. The EC20 of the active ingredient is 776 mg/L, the EC50 and the EC80 are > 1000 mg/L.

Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with the test item Alcohols, C6-8 alkyl-(even, linear), ethoxylated (2.5 EO)(batch number: ESD0006420) from 2012-08-22 to 2012-08-23, with the definitive exposure on 2012-08-23 at the Dr.U.Noack-Laboratorien, 31157 Sarstedt, Germany. Test system was activated sludge of the municipal treatment plant of 31137 Hildesheim, Germany. The test was carried out under static conditions with the substance concentrations 10 - 32 - 100 - 320 - 1000 mg/L. The respiration rates of the control, reference and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The mean inhibition of respiration for the test item replicates ranged from -19 % to 31 % .

In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate as reference item and the reference toxicity was determined. The EC₅₀-value for the reference item was 114 mg/L.

NOEC and EC-Values with Confidence Interval of Alcohols, C6-8 alkyl-(even, linear), ethoxylated (2.5 EO)

	EC-values [mg/L]	Confidence interval P = 95 [%] [mg/L]
NOEC*	320	–
EC10	618	575 - 661
EC20	776	724 - 851
EC50	> 1000	–
EC80	> 1000	–

The EC10 of Alcohols, C6-8 alkyl-(even, linear), ethoxylated (2.5 EO) is 618 mg/L. The test item is not toxic up to the concentration of 618 mg/L to activated sludge of a municipal sewage treatment plant. The EC50 is > 1000 mg/L.