Didecyldimethylammonium chloride

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-08-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 1 g/L was prepared and agitated.
- Eluate: Synthetic waste water acc. to OECD 209
- Differential loading: 1 - 3.2 - 10 - 32 - 100 mg/L
- Controls: Test medium without test or reference item.

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 3.9 g/L +/- 10 % was not necessary. The sludge was used within 24 h after sampling.
- Dry sludge concentration: 2.97 g/L corresponding to 1.49 g/L suspended solids in the test vessel.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

Not determined

Test temperature:

20.9 °C

pH:

7.99 (activated sludge)
7.55 (synthetic waste water)

Salinity:

None, freshwater

Details on test conditions:

TEST SYSTEM
- Test vessel: 1000 mL Erlenmeyer flask, DIN 12380
- Aeration: Permanent
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6 (3 at the beginning of the test and 3 at the end of the test)
- Composition of test medium:
Test concentration [mg a.i./L]
0.4 1.28 4.0 12.8 40.0
Inoculum 250
Synthetic waste water 16
Stock solution [mL] 0.5 1.6 5 16 50
Demineralised water 233.5 232.4 229 218 184

Reference item concentration [mg/L]
58 100 180
Inoculum 250
Synthetic waste water 16
Stock solution [µL] 96.7 166.7 300
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 transferring the solution to the measuring cell and the oxygen depletion was recorded for at least 3 minutes on a flat bed recorder.

TEST CONCENTRATIONS
- Range finding study
Inhibition of Respiration (Preliminary Tests)
Nominal Test Item Concentration Inhibition [%]
[mg/L]
10 16
100 95
1000 89

Reference substance (positive control):

yes

Remarks:

Copper (II) sulphate pentahydrate p.a.

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

ca. 5.95 other: mg a.i./L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: (5.22-6.73)

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

The EC-values of the test item were calculated by sigmoidal dose-response regression and the EC50-value of the reference item by linear regression using software GraphPadPrism. Calculations of the confidence intervals for the EC-values were carried out using standard procedures.

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]	[mg a.i./L]
	Control		1	32.4	21.7	–	–
			2	32.4	21.7
			3	32.4	21.7
			4	30.4	20.4
			5	28.8	19.3
			6	28.4	19.1
Test Item	1	0.4	1	32.0		-4	-4
			2	32.0		-4
			3	32.4		-5
			4	31.6		-3
			5	31.6		-3
	3.2	1.28	1	31.6		-3	-1
			2	30.8		0
			3	30.8		0
			4	31.2		-1
			5	31.2		-1
	10	4.0	1	25.6		17	6
			2	29.2		5
			3	32.0		-4
			4	30.0		3
			5	28.0		9
	32	12.8	1	24.4		21	33
			2	20.0		35
			3	19.6		36
			4	19.2		38
			5	19.6		36
	100	40.0	1	3.2		90	90
			2	3.2		90
			3	3.2		90
			4	2.8		91
			5	3.2		90
Reference Item	58	–	1	24.8		19	19
			2	24.8		19
			3	24.8		19
	100	–	4	16.8		45	45
			5	17.2		44
			2	16.8		45
	180	–	3	10.8		65	69
			4	10.4		66
			5	7.2		77

EC-Values with Confidence Interval of DDAC

	EC-values [mg a.i./L]	Confidence interval P = 95 [%] [mg a.i./L]
NOEC*	4.0	–
EC10	5.95	5.22 - 6.73
EC20	8.90	8.06 - 9.89
EC50	17.9	16.4 - 19.5

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the NOEC was 4.0 mg a.i. /L. The EC10 was 5.95 mg a.i. /L, the EC20 8.90 mg a.i./L and the EC50 17.9 mg a.i. /L.

Executive summary:

A respiration inhibition test with activated sludge was conducted according to OECD Guideline 209, in compliance with GLP. The test system was activated sludge of a municipal treatment plant. The test was conducted under static conditions with substance concentrations of 1.0, 3.2, 10, 32 and 100 mg/L, corresponding to 0.40, 1.28, 4.0, 12.8 and 40.0 mg a.i. /L. The respiration rates of the control, reference and test substance replicates were measured after a contact time of 3 h 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 -4 to 90%. 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 115 mg/L. Under the study conditions, the NOEC was 4.0 mg a.i. /L. The EC10 was 5.95 mg a.i. /L, the EC20 8.90 mg a.i./L and the EC50 17.9 mg a.i. /L (Fiebig, 2012). 

Description of key information

-         Key value from Registrant: 3 h EC50 = 17.9 mg a.i./L

-         Key value from DDAC biocides assessment report for Product Type 8 (June 2015): 21 d NOEC = 3 h EC50 = 14.3 mg a.i./L

In the present report, the 3 h EC50 of 14.3 mg a.i./L was selected as key value for the derivation of PNEC STP, in line with the methodology followed in the DDAC biocides assessment report for Product Type 8.

Key value for chemical safety assessment

EC50 for microorganisms:

14.3 mg/L

Additional information

A respiration inhibition test with activated sludge was conducted according to OECD Guideline 209, in compliance with GLP. The test system was activated sludge of a municipal treatment plant. The test was conducted under static conditions with substance concentrations of 1.0, 3.2, 10, 32 and 100 mg/L, corresponding to 0.40, 1.28, 4.0, 12.8 and 40.0 mg a.i. /L. The respiration rates of the control, reference and test substance replicates were measured after a contact time of 3 h 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 -4 to 90%. 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 115 mg/L. Under the study conditions, the NOEC was 4.0 mg a.i. /L. The EC10 was 5.95 mg a.i. /L, the EC20 8.90 mg a.i./L and the EC50 17.9 mg a.i. /L (Fiebig, 2012). 

The DDAC biocides assessment report for Product Type 8 conducted under Directive 98/8/EC (evaluating Competent Authority: Italy, June 2015, attached in Section 13 of the IUCLID dataset), reported an additional study on DDAC, apart from the above EQC study. This study resulted in a 3 h EC50 = 11.0 mg a.i./L. The RMS considered both studies as equally valid and therefore used a geomean value of 14.3 mg a.i./L as the endpoint for the product authorisation.