Fatty acids, tall-oil, 1-methyl-1,2-ethanediyl esters

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

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:

yes

Remarks:

(the pH reference substance stock solution was adjusted to 6.75, which is slightly outside the requested range of 7.5±0.5. Since the validity criterion regarding the reference substance was met the integrity or validity of the study was not affected.)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

not required

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance is poorly soluble in water. Weights of the test material were added directly to the test bottles followed by RO (reverse osmosis) water (284 mL), synthetic sewage (16 mL) and antifoam agent (silicone oil in water, 100 µL). Immediately prior to initiation of the respirometer, the required volume of activated sludge (200 mL) was added to each bottle.
- Controls: RO water, synthetic sewage and antifoam agent alone.

The final volume of antifoam was not considered significant and was not accounted for in the final overall volume (500 mL).

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Preparation of inoculum for exposure: Activated sludge was obtained the day before the start of the test. The mixed liquor suspended solids (MLSS) content was determined, synthetic sewage (50 mL/L) was added and the mixture was aerated overnight. On the day of the test, the MLSS content of the sludge was determined and adjusted to 4 g/L by the addition of tap water.
- Pretreatment: none
- Initial biomass concentration: 4 g MLSS/L

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

21°C ± 0.4°C

pH:

test start: 7.4-7.7 (adjusted using 0.1M NaOH as required)
test end: 7.8-8.0

Dissolved oxygen:

3.7 - 6.6 mg O2/L (initial measured dissolved oxygen concentration in the cultures)

Nominal and measured concentrations:

nominal 10, 100, 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test instrument: automated respirometer and associated software (Coordinated Environmental System (CES).
- Test vessel: 500 mL bottles fitted with an oxygen probe, a sinter to deliver air and a nitrogen gas nozzle
- Aeration: oil-free compressed air at 1 L/min
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 5
- Biomass loading rate: 1.6 g/L (MLSS)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis (RO) water

OTHER TEST CONDITIONS
- Adjustment of pH: to 7.0-8.0 with 0.1M NaOH as required

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : oxygen concentration after 3 hours contact time

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study/limit test
- Test concentrations: 10, 100, 1000 mg/L
- Results used to determine the conditions for the definitive study: As there was no significant inhibition observed in any test groups, the study was reported as a limit test.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

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

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

Remarks:

respiration rate

Details on results:

The mean inhibition observed in cultures with the test substance ranged between -11 and -2% (i.e. slight stimulation) with ranges of -26 to 30% (coefficient of variation 7 - 19%; see Table 1).

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (3 h): 11.4 mg/L (95% confidence limits 2.7-47.3 mg/L)

Reported statistics and error estimates:

EC calculation: A logistic sigmoid curve was fitted to the data by Marquardt's iterative compromise method.
NOEC calculation: The F1 approximate test for monotonicity of dose-response was applied. Since it was not significant at the 1% level, the Williams' test was performed.
Test and reference substance were analysed separately.
Data were analysed using SAS 9.1.3, Proc NLIN (SAS Institute, 2002).

Table 1 Specific respiration rates, inhibition and coefficient of variation

 

Group	Nominal Conc. (mg/L)	Bottle No.	Specific respiration rate (Rs mgO2/gh)	Mean Rs (mgO2/gh)	% inhibition	Mean % inhibition	Coefficient of variation (%)
Control	-	1	31.0	26.7	-	-	23
		2	28.7
		3	29.2
		4	28.7
		5	15.8
Test Substance	10	6	26.4	29.4	1	-10	7
		7	29.0		-9
		8	29.4		-10
		9	31.2		-17
		10	31.3		-17
	100	11	26.8	27.2	0	-2	19
		12	18.8		30
		13	27.9		-5
		14	33.1		-24
		15	29.6		-11
	1000	16	32.7	29.6	-23	-11	13
		17	29.6		-11
		18	33.6		-26
		19	24.0		10
		20	28.1		-5

The data presented were calculated using unrounded values stored in the computer database. Minor numerical differences may be observed in the respiration rate calculation if rounded values are used to calculate the data. This minor discrepancy is not considered to be significant

Negative inhibition indicates a respiration rate greater than that of the mean control respiration rate

Validity criteria fulfilled:

yes

Remarks:

The mean specific respiration rate of the blank controls was ≥20 mgO2/gh with a coefficient of variation of ≤30%. The EC50 of 3,5-dichlorophenol was in the accepted range of 2-25 mg/L.

Description of key information

Toxicity to microorganisms: no inhibition of respiration rates (OECD 209)

Additional information

EC50 for microorganisms > 1000 mg/L
EC10 or NOEC for microorganisms ≥ 1000 mg/L

The results indicate that the test material is not toxic to sewage bacteria up to the limits of its water solubility (<1 mg/L) and a NOEC and thus a PNEC cannot be determined.