Octadecanoic acid, reaction products with diethylenetriamine and urea, acetates

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:

2013

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 (incl. QA statement)

Specific details on test material used for the study:

Identification: FAT #: 93580/A
Batch: 0022357000
Purity: 94.3 %
Expiry date: 15 December 2015
Description at Room Temperature: White-yellowish waxy solid
Storage Conditions: Room temperature at 20 ± 5 °C, in the dark.

Analytical monitoring:

not specified

Details on sampling:

No data

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Based on the sponsor’s information, no stock solution could be prepared due to the low water solubility of the test item.
Therefore, test item amounts of 5.02, 50.47, 500.88, 500.43 and 500.92 mg were weighed by means of an analytical balance and transferred to the designated test vessels with 234 mL water.
The test item was mixed into the water by intense stirring for 10 minutes at room temperature to obtain a homogeneous dispersion of the test item. No emulsifiers or solvents were used.
After stirring, the appearance of the test media was evaluated and the pH measured. All loading rates were clearly above the water solubility limit of FAT# 93580/A under the test conditions. The pH of the dispersions was adjusted from pH 3.6 - 6.3 to pH 7.7 - 7.9 with a sodium hydroxide solution.
Then, 16 mL of synthetic sewage feed and 250 mL of the activated sludge inoculum were added

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

The study was performed with aerobic activated sludge from a waste-water treatment plant (ARA Ergolz II, Füllinsdorf / Switzerland) treating predominantly domestic wastewater.

The sludge was washed twice with chlorine free tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.

Based on this ratio, a calculated amount of wet sludge was resuspended in tap water to obtain a concentration equivalent to 3 g dry material per liter. During the holding period of one day prior to use, the sludge was fed once with 50 mL synthetic sewage feed (see Section 3.3) per liter and was kept at room temperature under continuous aeration until use. Before use, the dry weight of the activated sludge was measured again and found to be 3 g dry weight per liter in the inoculum used for the test, equivalent to 1.5 g/L in the final incubation mixture. The pH of the activated sludge inoculum was adjusted from 6.8 to 7.5 with a 1M NaOH solution.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

Study terminated after 3 h

Hardness:

No data

Test temperature:

20 - 21 °C

pH:

7-8

Dissolved oxygen:

≥7.8 mg/L

Salinity:

No data

Nominal and measured concentrations:

Nominal concentrations: 10, 100 and 1000 mg/L

Details on test conditions:

During the incubation period of 3 hours all vessels were continuously aerated by intense stirring on magnetic stirrers to avoid possible foaming and/or stripping of the test item. The concentration of dissolved oxygen did not drop below 60-70 % saturation and the sludge flocs were maintained in suspension during the incubation period. Just before measurement of the respiration rates, at the end of the incubation period, the dissolved oxygen concentrations were at least 7.9 mg/L. The temperature in the test media, measured in one control, was 21 °C at the start and 20 °C at the end of the incubation period.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

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:

Based on the physical appearance of the test suspensions, the saturation concentration (water solubility limit of FAT #: 93580/A under the test conditions) was reached at a concentration of <10 mg/L.

At all tested loading rates up to and including 1000 mg/L, the inhibition of the respiration rate of activated sludge after the incubation period of three hours was in the range 1.5 to 7.0 % compared to the control. For the three replicates of the highest test loading rate of 1000 mg/L the mean inhibition of the respiration rate was calculated to be 5.6 %, compared to the control. According to the results of a Student t-test (one sided smaller, α = 0.05), the test item FAT #: 93580/A had no statistically significant inhibitory effect on the respiration rate of up to and including the highest test item loading rate of 1000 mg/L.

Thus, the 3-hour NOEC of FAT #: 93580/A to activated sludge micro-organisms was at the saturation concentration of the test item under the test conditions, or at least 1000 mg/L. This value might even be higher, but loading rates exceeding 1000 mg/L were not tested. The 3-hour EC10, EC20, EC50 and EC80 could not be calculated, but were clearly higher than 1000 mg/L.

In conformity with the guideline, a definitive test was not necessary since in the range-finding test the test item showed no statistically significant inhibition of the respiration rate at the highest tested concentration (performed as triplicate).

Results with reference substance (positive control):

The 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was calculated to be 4.0 mg/L with 95 % confidence limits from 1.5 to 6.2 mg/L. The 3-hour EC50 is within the guideline-recommended range of 2–25 mg/L, confirming suitability of the activated sludge used.

Reported statistics and error estimates:

The NOEC was derived by statistics using the Student t-test (Sachs, 1984). Compared were the three replicates of the highest test item loading rate with the four blank control replicates using one-tailed (smaller) hypothesis and α = 0.05.

The 3-hour EC50 of the reference item 3,5-dichlorophenol was calculated by Weibull Analysis (Christensen, 1984 a, b).

Respiration Rates of the Blank Controls:

The specific respiration rates for the two blank control replicates prepared at the beginning of the exposure period were 40 and 42 mg O₂ per dry weight of activated sludge per hour and for the two blank controls prepared at the end of the test were 33 and 39 mg O₂/gh. All four values are higher than the 20 mg O₂/gh stipulated in the guideline. The resulting coefficient of variation of oxygen uptake rate in the control replicates has a value of 10 % and is therefore, as stipulated in the guideline, not more than 30 %.

Validity of the Test:

The test is regarded as valid since: - the specific respiration rate of the blank controls was in the range of 33 to 42 mg oxygen per gram dry weight of sludge per hour and therefore, higher than 20 mg O₂/gh.

- the coefficient of variation of oxygen uptake rate in control replicates was 10 % and therefore, less than 30 %

- the EC50 (3-Hour) of the reference item 3,5-dichlorophenol was 4 mg/L and therefore, in the range 2 to 25 mg/L

Influence of FAT #: 93580/A (Test Item) and3,5-Dichlorophenol(ReferenceItem) on the Oxygen Consumption of Activated Sludge

Vessel no.	Nominal concentration of test chemical [mg/L]	Oxygen consumption rate		Inhibition   %	pH values		Oxygen concentration [mgO2/L]
		Respiration rate ( R) [mg O2/Lh]	Specific respiration rate (RS) [mg O2/gh]
					start*	end*	start*	end*
Control
1	0	60.60	40.40		7.3	7.8	7.7	8.5
2	0	62.64	41.76		7.2	7.8	7.8	8.6
11	0	49.56	33.04		7.2	7.9	7.9	8.1
12	0	58.95	39.30		7.2	7.7	8.0	8.2
Mean		57.94	38.63
Standard Deviation		5.79	3.86
Coefficient of Variation		9.99%	9.99%
Test item
6	10	57.07	38.04	1.5	7.2	7.7	7.8	8.1
7	100	55.54	37.03	4.1	7.2	7.7	7.9	8.0
8	1000	56.27	37.51	2.9	7.4	7.7	7.9	8.2
9	1000	54.00	36.00	6.8	7.3	7.7	7.8	8.2
10	1000	53.89	35.93	7.0	7.2	7.8	7.8	8.1
Reference item
3	3.2	31.33	20.88	45.9	7.1	7.9	7.6	8.6
4	10	17.57	11.71	69.7	7.1	7.8	7.8	7.9
5	32	4.46	2.97	92.3	7.4	7.9	7.9	8.3

Validity criteria fulfilled:

yes

Conclusions:

The 3-hour NOEC of FAT #: 93580/A to activated sludge micro-organisms was at least 1000 mg/L. This value might even be higher, but loading rates exceeding 1000 mg/L were not tested. The 3-hour EC10, EC20, EC50 and EC80 could not be calculated, but were clearly higher than 1000 mg/L.

Executive summary:

The inhibitory effect of the test item FAT #: 93580/A on the respiration rate of aerobic wastewater micro-organisms of activated sludge was investigated in a 3-hour respiration inhibition test according to the OECD Guideline for Testing of Chemicals, No. 209, adopted 2010.

A range-finding test using the following three test item loading rates 10 mg/L, 100 mg/L and 1000 mg/L was performed. Single test vessel replicates were tested for the two lower loading rates, whereas for the highest test item loading rate three replicates were prepared.

As a positive control, the reference item 3,5-dichlorophenol was tested under identical test conditions, at concentrations of 3.2, 10 and 32 mg/L (all single replicates).

Four blank controls, containing an equal volume of activated sludge and synthetic medium but no test or reference item, were included in the range-finding test.

The results of the blank and positive control treatments confirmed suitability of the activated sludge and the method used. The validity criteria were fulfilled.

The test item FAT #: 93580/A had no statistically significant inhibitory effect on the respiration rate of activated sludge at all tested loading rates up to and including 1000 mg/L after the incubation period of three hours.

Thus, the 3-hour NOEC of FAT #: 93580/A to activated sludge micro-organisms was at least 1000 mg/L. This value might even be higher, but loading rates exceeding 1000 mg/L were not tested. The 3-hour EC10, EC20, EC50 and EC80 could not be calculated, but were clearly higher than 1000 mg/L.

The results can be summarized as follows:

EC10 > 1000 mg/L

EC20: > 1000 mg/L

EC50: > 1000 mg/L

EC80: > 1000 mg/L

NOEC: ≥ 1000 mg/L

In conformity with the guideline, a definitive test was not necessary since in the range-finding test the test item showed no statistically significant inhibition of the respiration rate at the highest tested concentration (performed as triplicate).

Description of key information

The inhibitory effect of the test item FAT #: 93580/A on the respiration rate of aerobic wastewater micro-organisms of activated sludge was investigated in a 3-hour respiration inhibition test according to the OECD Guideline for Testing of Chemicals, No. 209, adopted 2010.

A range-finding test using the following three test item loading rates 10 mg/L, 100 mg/L and 1000 mg/L was performed. Single test vessel replicates were tested for the two lower loading rates, whereas for the highest test item loading rate three replicates were prepared.

As a positive control, the reference item 3,5-dichlorophenol was tested under identical test conditions, at concentrations of 3.2, 10 and 32 mg/L (all single replicates).

Four blank controls, containing an equal volume of activated sludge and synthetic medium but no test or reference item, were included in the range-finding test.

The results of the blank and positive control treatments confirmed suitability of the activated sludge and the method used. The validity criteria were fulfilled.

The test item FAT #: 93580/A had no statistically significant inhibitory effect on the respiration rate of activated sludge at all tested loading rates up to and including 1000 mg/L after the incubation period of three hours.

Thus, the 3-hour NOEC of FAT #: 93580/A to activated sludge micro-organisms was at least 1000 mg/L. This value might even be higher, but loading rates exceeding 1000 mg/L were not tested. The 3-hour EC10, EC20, EC50 and EC80 could not be calculated, but were clearly higher than 1000 mg/L.

The results can be summarized as follows:

EC10 > 1000 mg/L

EC20: > 1000 mg/L

EC50: > 1000 mg/L

EC80: > 1000 mg/L

NOEC: ≥ 1000 mg/L

In conformity with the guideline, a definitive test was not necessary since in the range-finding test the test item showed no statistically significant inhibition of the respiration rate at the highest tested concentration (performed as triplicate).

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L