Octadecanoic acid, reaction products with triethylenetetramine

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:

2017-05-22 to 2017-05-23

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

Version / remarks:

2010-07-22

Deviations:

no

Principles of method if other than guideline:

In the preliminary test, additional replicates were prepared to determine the inhibition of nitrification by measuring with addition of Allylthiourea.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

not applicable (no analytics)

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: weighed out directly in the Erlenmeyer flasks

- Eluate: n.a.
- Differential loading: no
- Controls: reference item concentrations were diluted out of a stock solution.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): n.a.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): n.a.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): not reported

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: no
- Name and location of sewage treatment plant where inoculum was collected:
municipal treatment plant of 31137 Hildesheim, Germany

- Method of cultivation: n.a.
- Preparation of inoculum for exposure:

- Pretreatment:
The sludge was washed twice with chlorine free tap water and adjusted to a dry sludge concentration of 3.0 g/L ± 10 %. The sludge was used within 24 h after sampling.

- Initial biomass concentration:
Dry sludge concentration 3.30 g/L (corresponding to 1.65 g/L suspended solids in the test vessels)

Test type:

static

Water media type:

freshwater

Remarks:

Synthetic waste water according to OECD Guideline 209

Limit test:

yes

Total exposure duration:

3 h

Hardness:

not reported

Test temperature:

actually measured 21.6 °C

pH:

activated sludge 7.49
sytnthetic waste water 7.34
test solution 7.66 (1000mg/l)

Dissolved oxygen:

3 replicates with 6.46, 7.12, 7.16 [mg O2/L] determined at the start of the measurement

Salinity:

not reported

Conductivity:

not reported

Nominal and measured concentrations:

preliminary test: 10, 100, 1000 mg/L
main test: 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flasks, DIN 12380
- Material, size, headspace, fill volume: glas, 500 mL, 250 mL
- Aeration: yes
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 3
- No. of vessels per abiotic control (replicates): n.a.
- Sludge concentration (weight of dry solids per volume): Dry sludge concentration 3.30 g/L (corresponding to 1.65 g/L suspended solids in the test vessels)
- Weight of dry solids per volume of reaction mixture per unit of time: ?
- Nutrients provided for bacteria: Synthetic waste water according to OECD-Guideline 209
- Nitrification inhibitor used (delete if not applicable): N-allylthiourea
- Biomass loading rate: see above

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: demineralised water
- Particulate matter: n.a.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: not reported
- Light intensity: n.a.
- Details on termination of incubation: not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
TEST CONCENTRATIONS
- Spacing factor for test concentrations: preliminary test: 10; main test: limit test
- Justification for using fewer concentrations than requested by guideline:
Based on the results of the preliminary test it could concluded that the heterotrophic respiration as well as the nitrification are not inhibited. Therefore, the definitive study was carried out with the concentration of 1000 mg/L in 3 replicates (limit test to determine the NOEC).

- Range finding study: yes
- Test concentrations: 10, 100, 1000 mg/l
- Results used to determine the conditions for the definitive study: no significant inhibition of respiration or nitrification in the definitive study was observed, even the inhibitions of the heterotrophic respiration or nitrification show no appreciable value.

Reference substance (positive control):

yes

Remarks:

Copper (II) sulphate pentahydrate

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: not reported

- Effect concentrations exceeding solubility of substance in test medium:

- Adsorption (e.g. of test material to the walls of the test container): not measured / reported

- Blank controls oxygen uptake rate: 25.3 mg mg O2/g x h

- Coefficient of variation of oxygen uptake rate in control replicates: 5.3%

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels or copper (II) sulphate pentahydrate: EC50=99.5 mg/L (95 % Confidence Interval = 95.7 - 104 mg/L)
- Other: recommended range of validity of 53 - 155 mg/L

Reported statistics and error estimates:

Statistical Analysis (NOEC)

One Way Analysis of Variance

Normality Test (Shapiro-Wilk) Failed (P < 0.050)

Equal Variance Test: Passed (P = 0.566)

Group Name N Missing Mean Std Dev SEM
Control 6 0 41.683 2.427 0.991
1000 mg/L 3 0 43.667 0.737 0.426

Source of Variation DF SS MS F P
Between Groups 1 7.867 7.867 1.804 0.221
Residual 7 30.535 4.362
Total 8 38.402

The differences in the mean values among the treatment groups are not great enough to exclude the possibility that the difference is due to random sampling variability; there is not a statistically significant difference (P = 0.221).

Power of performed test with alpha = 0.050: 0.117

The power of the performed test (0.117) is below the desired power of 0.800.
Less than desired power indicates you are less likely to detect a difference when one actually exists. Negative results should be interpreted cautiously.

 Preliminary Test (non GLP)

 

Inhibitions determined in the preliminary test are listed in Table 4 and Table 5.

 

Table4:        Inhibitions in the Preliminary Test

Nominal Test Item Concentration [mg/L]	pH (test item in medium)	Inhibition [%]
1^stpreliminary test - without pH adjustment, direct aeration
10	-	-5
	-	-5
100	-	-4
	-	-4
1000	7.66	-5
	-	1

Table5:        Inhibitions in the Preliminary Test (with / without ATU)

Nominal Test Item Concentration [mg/L]	Inhibition of total respiration [%]	Inhibition of heterotrophic respiration [%]	Inhibition of nitrification [%]
1000	-5	1	-11
	1	-4	4

Oxygen Uptake Rates, Specific Respiration Rates and Inhibition of the Respiration

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 to the oxygen uptake rates of the control. The specific respiration rates are listed inTable 6. The results are listed in Table 7.

Table6:      Specific Respiration Rates of the Control Replicates

		Repl.	Specific Respiration Rate Rs [mg O2/g×h]
	Control	1	25.2
		2	25.9
		3	22.4
		4	26.4
		5	26.3
		6	25.3
Mean value			25.3

Repl. = Replicate

Suspended solids in the test vessel: 1.65 g/L

Table7:    Oxygen Uptake Rates, Inhibition of the Respiration of Control, Test and Reference Item Concentrations

Test and Reference Item Concentration [mg/L]	Repl.	Oxygen Uptake Rate R [mg O2/L×h]	Inhibition [%]	Mean Inhibition [%]
Control	1	41.6	-	-
	2	42.8
	3	37.0
	4	43.5
	5	43.4
	6	41.8
Test item 1000	1	43.1	-3	-4
	2	44.5	-6
	3	43.4	-3
Reference item 58	1	31.7	24	26
	2	31.5	24
	3	29.6	29
100	1	22.3	47	48
	2	21.8	48
	3	20.7	50
180	1	8.7	79	79
	2	9.3	78
	3	8.7	79

Repl. = Replicate       

Coefficient of variation of oxygen uptake rates of the control: 5.3%

Suspended solids in the test vessel: 1.65 g/L

Validity criteria fulfilled:

yes

Conclusions:

The NOEC of the test item Octadecanoic acid, reaction products with triethylenetetramine is ≥ 1000 mg/L.

Executive summary:

For the test item Octadecanoic acid, reaction products with triethylenetetramine a respiration inhibition test with activated sludge according to OECD Guideline No. 209 (22 July 2010) was carried out.

A preliminary test performed with test item concentrations of 10, 100, 1000 mg/L showed no relevant inhibition of the heterotrophic respiration or nitrification (tested with Allylthiourea ATU).

The main test was performed as a limit test with a duration of 3 h. The NOEC resulted to be ≥ 1000 mg/L.

Description of key information

For Octadecanoic acid, reaction products with triethylenetetramine a respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 (2010) was carried out.

A preliminary test with 10, 100, 1000 mg/l showed no relevant inhibiting of the heterotrophic respiration or nitrification (tested with Allylurea ATU).

The main test was made as limit test resulting in a 3h-NOEC=1000 mg/l.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L