Fatty acids, coco, esters with 3,3'-oxybis[1,2-propanediol]

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:

22 JUN 2016 to 23 JUN 2016

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 (Carbon and Ammonium Oxidation))

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

Incubation Period: 3 hours

Vehicle:

no

Details on test solutions:

Dosage of Test Item: Appropriate amounts of test item were directly weighed into the test vessels and test water was added. The composition was stirred intensively.
Test Concentrations: Nominal 10, 32, 100, 320 and 1000 mg test item/L
Control: Six controls (pure water, synthetic sewage and inoculum, but without addition of the test item) were tested in parallel.

Stock Solution of 3,5-Dichlorophenol: In parallel to the study with the test item, the reference item 3,5-dichlorophenol was tested at the nominal test concentrations of 1, 4, and 16 mg/L (five replicates each test concentration) under otherwise identical test conditions. A stock solution of 3,5-dichlorophenol was prepared accord-ing to the OECD Guideline No. 209: 1 g of 3,5-dichloro-phenol was dissolved in 1000 mL pure water. Warm water was used to accelerate the dissolution. The solution was filled up to volume when it had cooled to room temperature. The final pH was 7.3 and therefore in the range of 7 to 8. NaOH was used for the adjustment of the pH.

Stock Solution of N-allylthiourea (ATU): In parallel a nitrification inhibitor N-allylthiourea (ATU) was tested in the same way with six separate controls and at the identical nominal concentrations of the test item and also the reference item 3,5-dichlorophenol under otherwise identical test conditions. A stock solution of N-allylthiourea was prepared according to the OECD Guideline No. 209: a stock solution of 2.32 g/L N-allylthiourea was prepared. 2.5 mL of this stock solution were added to an incubation mixture of final volume of 500 mL. This resulted in a final concentration of 11.6 mg ATU/L.

Test organisms (species):

activated sludge

Details on inoculum:

Species / Origin: Activated sludge, microorganisms from a domestic waste water treatment plant was supplied by the municipal sewage treatment plant Bensheim, Germany.
Conditioning: The activated sludge used for this study was used as collect-ed, but coarse particles were removed by settling for a short period (20 minutes) and then the upper layer decanted. During holding prior to use the sludge was fed with 50 mL synthetic sewage (see below) per litre and kept aerated at room temperature overnight. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight determined. Based on the sludge dry matter, calculated amounts of wet sludge were suspended in pure water to yield a concentra-tion equivalent to about 3.0 g/L on dry weight basis. This level gives a concentration of 1.2 g/L suspended solids in the test medium. The pH of the activated sludge inoculum was 7.2 and therefore no adjustment necessary.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Remarks on exposure duration:

For the measurement of the respiration rate a sample of each test medium was poured into a Karlsruher flask after exactly 3 hours incubation time. The oxygen con-centration was measured with an oxygen electrode and recorded for about ten minutes.

Test temperature:

20°C ± 2°C

pH:

6.6 to 7.8

Dissolved oxygen:

5.1 to 8.6

Nominal and measured concentrations:

Test Concentrations: 10, 32, 100, 320 and 1000 mg test item/L; 1, 4 and 16 mg 3,5-Dichlorophenol/L and six inoculum controls

Details on test conditions:

Surrounding Type: Controlled environment room
Temperature: 20°C ± 2°C during pre-incubation (3 hours) and evaluation period
Aeration: Compressed air (1.017 litre per minute)

Reference substance (positive control):

yes

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

146.06 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

597.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

LOEC

Effect conc.:

320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

842.11 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

1 012.16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Duration:

3 h

Dose descriptor:

LOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

99.63 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of respiration due to nitrification

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

201.08 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of respiration due to nitrification

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of respiration due to nitrification

Duration:

3 h

Dose descriptor:

LOEC

Effect conc.:

320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of respiration due to nitrification

Details on results:

Inhibition of Respiration Rate (Total Respiration):
The influence of the test item on the respiration rate of activated sludge is presented in Table 3 and in Figure 1.
Total respiration:
In comparison to the inoculum controls, the total respiration rate of the activated sludge was not inhibited at test concentrations up to 100 mg/L (inhibition below 10%) and moderately inhibited for the two highest test concentrations. The inhibition was 6.5%, -6.1% and -6.5% for the test concentrations of 10 mg/L, 32 mg/L and 100 mg/l, respec-tively. For the test item concentrations of 320 mg/L and 1000 mg/L, the inhibition rates were 34.4%, and 64.2%, respectively. Concentrations exceeding 1000 mg/L nominal were not tested.

3-hour EC10 and 3-hour EC50 based on Total Respiration:
The 3-hour EC10 and EC50 for total respiration were established to be 146.06 mg/L (95% confidence limits (cl): lower cl: 125.78 mg/L, higher cl: 169.61 mg/L) and 597.1 mg/L (95% confidence limits (cl): lower cl: 492.13 mg/L, higher cl: 718.86 mg/L), respectively; the NOEC was determined to be at a test item concentration of 100 mg/L, the LOEC at a test item concentration of 320 mg/L.

Inhibition of Respiration Rate (Heterotrophic Respiration without Nitrification):
The influence of the test item on the respiration rate of activated sludge is presented in Table 4 and in Figure 2.
In comparison to the inoculum controls the respiration was not inhibited for test item concentrations up to 320 mg/L and moderately inhibited at the highest test item concen-tration. The inhibition rates were 2.9%, 2.1%, -20.0% and
-6.8% for test item concentrations of 10 mg/L, 32 mg/L, 100 mg/L and 320 mg/L, respectively. At a concentration of 1000 mg/L, the inhibition of the respiration rate was 44.4%.

3-hour EC10 and 3-hour EC50 based on Heterotrophic Respiration without Nitrification:
The 3-hour EC10 and EC50 for heterotrophic respiration were established to be 842.11 mg/L (95% confidence limits (cl): lower cl: 0.00 mg/L, higher cl: n.d.) and 1012.16 mg/L (95% confidence limits (cl): lower cl: n.d., higher cl: n.d.), respectively; the NOEC was determined to be at a test item concentration of 320 mg/L, the LOEC at a test item concen-tration of 1000 mg/L.
n.d.: not determined due to mathematical reasons
Inhibition of Respiration Rate based on Nitrification:

In comparison to the inoculum controls, the respiration rates of the activated sludge based on nitrification were almost not inhibited at test concentrations up to 100 mg/L (inhibition below 11%) and strongly inhibited for the two highest test concentrations. The inhibition was 10.5%,
-15.2% and 8.5% for the test concentrations of 10 mg/L, 32 mg/L and 100 mg/l, respectively. For the test item concentrations of 320 mg/L and 1000 mg/L, the inhibition rates were 80.3%, and 86.2%, respectively. Concentrations exceeding 1000 mg/L nominal were not tested.

3-hour EC10 and 3-hour EC50 based on Oxygen Uptake due to Nitrification:
For the oxygen uptake due to nitrification the 3-hour EC10 and EC50 were established to be 99.63 mg/L (95% confidence limits (cl): lower cl: 57.75 mg/L, higher cl: 171.88 mg/L) and 201.08 mg/L (95% confidence limits (cl): lower cl: 110.45 mg/L, higher cl: 375.03 mg/L), respectively; the NOEC was determined to be at a test item concentration of 100 mg/L, the LOEC at a test item concentration of 320 mg/L.

Results with reference substance (positive control):

Inhibition of Respiration Rate of Total Respiration:
The inhibition of the activated sludge treated with the reference item was in the range of 38% to 83% for the test concentrations from 1 until 16 mg/L. The results are summarized in Table 3 and in Figure 1.
3-hour EC10, EC20 and EC50 of 3,5-Dichlorophenol of Total Respiration:
The 3-hour EC10, EC20 and EC50 and their 95 % confidence limits of 3,5-Dichlorophenol were calculated. The results are listed below:
Parameter EC10 EC20 EC50
Value [mg/l] 0.13 0.36 2.44
lower 95%-cl 0.04 0.11 0.57
upper 95%-cl 0.47 1.20 10.35


Inhibition of Respiration Rate of Heterotrophic Respiration without Nitrification:
The inhibition of the activated sludge treated with the reference item was in the range of 11% to 70% for the test concentrations from 1 until 16 mg/L. The results are sum-marized in Table 4 and in Figure 2.
3-hour EC10, EC20 and EC50 of 3,5-Dichlorophenol of Heterotrophic Respiration:
The 3-hour EC10, EC20 and EC50 and their 95 % confidence limits of 3,5-Dichlorophenol were calculated. The results are listed below:
Parameter EC10 EC20 EC50
Value [mg/l] 3.19 4.78 10.33
lower 95%-cl 1.76 2.72 5.19
upper 95%-cl 5.80 8.38 20.33


Inhibition of Respiration Rate of Oxygen Uptake due to Nitrification:
The inhibition of the activated sludge treated with the ref-erence item was in the range of 68% to 98% for the test concentrations from 1 until 16 mg/L. The results are sum-marized in Table 5.
 
3-hour EC10, EC20 and EC50 of 3,5-Dichlorophenol of Oxygen Uptake due to Nitrification: Based on measured inhibition rates, the 3-hour EC10, EC20 and EC50 and their 95% confidence limits were determined. The results are listed below:
Parameter EC10 EC20 EC50
Value [mg/l] 0.09 0.17 0.53
lower 95%-cl 0.00 0.01 0.01
upper 95%-cl 3.81 5.28 34.57

Reported statistics and error estimates:

Statistical Analysis: For normal distribution of the data, the Shapiro-Wilk`s Test was used, the variance homogeneity was tested by the Leven`s Test. Subsequently, the Williams Multiple Sequential t-test Procedure (one-sided smaller) was used to determine the LOEC and NOEC values, if possible. The 3-hour ECx and their 95 %-confidence limits were determined via nonlinear regression using the Levenberg-Marquardt optimization. The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ® ToxRat Solutions GmbH.

Influence of the test item and the Reference Item 3,5-Dichlorophenol on Total Oxygen Consumption of Activated Sludge

Flask No.	Treatment	Concentration	Oxygen consumption [mean]^1	Coefficient of Variation [mean]^1	Inhibition [mean]^1	pH-values [mean]^1		Oxygen concentration [mean][mg O2/L]^1
		[mg/L]	[mg O2/L h]	%	%	start*	end*	start*	end*
1, 2, 18, 19, 45,46	control	---	25.8	14.3	---	6.9	7.3	5.8	7.6
3-7	3.5-DCP	1	16.1	---	37.7	7.1	7.6	6.1	7.6
8-12	3.5-DCP	4	12.0	---	53.5	7.1	7.8	5.6	8.2
13-17	3.5-DCP	16	4.3	---	83.3	7.1	7.3	7.0	8.4
20-24	Test item	10	24.1	---	6.5	6.9	7.3	5.6	7.8
25-29	Test item	32	27.4	---	-6.1	7.1	7.2	5.7	6.2
30-34	Test item	100	27.5	---	-6.5	7.0	7.2	6.0	6.6
35-39	Test item	320	16.9	---	34.4	6.8	6.8	7.7	7.9
40-44	Test item	1000	9.2	---	64.2	6.9	6.6	7.1	8.1
^1: mean value of 5 replicates, 6 replicates in case of control
*: Start and end of 3-hour aeration
DCP:	3,5-Dichlorophenol
CV:	standard deviation / mean * 100

 

Influence of the Test Item and the Reference Item 3,5-Dichlorophenol on Heterotrophic Oxygen Consumption of Activated Sludge (nitrification respiration inhibited by ATU)

Flask No.	Treatment	Concentration	Oxygen consumption [mean]^1	Coefficient of Variation [mean]^1	Inhibition [mean]^1	pH-values [mean]^1		Oxygen concentration [mean][mg O2/L]^1
		[mg/L]	[mg O2/L h]	%	%	start*	end*	start*	end*
47, 48, 64, 65, 91, 92	control	---	13.6	16.9	---	7.0	7.8	5.9	8.1
49-53	3.5-DCP	1	12.1	---	10.9	7.1	7.8	6.0	8.1
54-58	3.5-DCP	4	11.2	---	17.9	7.1	7.7	5.9	8.2
59-63	3.5-DCP	16	4.1	---	70.0	7.1	7.3	6.8	8.6
66-70	Test item	10	13.2	---	2.9	6.9	7.7	5.1	8.0
71-75	Test item	32	13.3	---	2.1	7.1	7.8	5.3	7.2
76-80	Test item	100	16.3	---	-20.0	7.0	7.7	5.6	7.2
81-85	Test item	320	14.5	---	-6.8	6.9	7.5	7.6	8.0
86-90	Test item	1000	7.6	---	44.4	6.9	7.1	6.8	8.2
^1: mean value of 5 replicates, 6 replicates in case of control
*: Start and end of 3-hour aeration
DCP:	3,5-Dichlorophenol
CV:	standard deviation / mean * 100

Influence of the Test Item and the Reference Item 3,5-Dichlorophenol on Oxygen Consumption based on Nitrification of Activated Sludge

Treatment	Concentration [mg/L]	Oxygen consumption [mean]^1[mg O2/L h]	Coefficient of Variation [mean]^1%	Inhibition [mean]^1%
control	---	12.2	12.3	---
3.5-DCP	1	4.0	---	67.5
3.5-DCP	4	0.8	---	93.1
3.5-DCP	16	0.2	---	98.0
Test item	10	10.9	---	10.5
Test item	32	14.1	---	-15.2
Test item	100	11.2	---	8.5
Test item	320	2.4	---	80.3
Test item	1000	1.7	---	86.2
^1: mean value of 5 replicates, 6 replicates in case of control
*: Start and end of 3-hour aeration
DCP:	3,5-Dichlorophenol
CV:	standard deviation / mean * 100

Validity Criteria of the Study

Inoculum Control:	The oxygen uptake in the blank controls was 21.5 mg/g/h and therefore clearly above 20 mg oxygen/g of activated sludge (dry weight of suspended solids) in an hour. The respiration rates of the six controls differed not more than 30%. The value was 14.3% for the total respiration and 16.9% for the heterotrophic respiration. The validity criterion was fulfilled.
Reference Item (Total Respiration):	The 3-hour EC50 of the reference item 3,5-dichlorophenol for the total respiration was 2.44 mg/L and therefore in the range of 2 to 25 mg/L for the used activated sludge batch.
Reference Item (Heterotrophic Respiration):	The 3-hour EC50 of the reference item 3,5-dichlorophenol for the heterotrophic respiration without nitrification was 10.33 mg/L and therefore also in the range of 5 to 40 mg/L for the used activated sludge batch.
Reference Item (Nitrification):	The 3-hour EC50 of the reference item 3,5-dichlorophenol for the oxygen uptake due to nitrification was 0.53 mg/L and therefore in the range of 0.1 to 10 mg/L for the used activated sludge batch.

Validity criteria fulfilled:

yes

Conclusions:

3h-EC50 (activated sludge, total respiration inhibition) = 597.1 mg/L; 3h-EC10 (activated sludge, total respiration inhibition) = 146.06 mg/L; NOEC (activated sludge, total respiration inhibition) = 100 mg/L

Executive summary:

The influence of the test item on the activity of activated sludge was evaluated by measuring the respiration rate under defined conditions. The respiration rate (oxygen consumption) of an aerobic activated sludge fed with a standard amount of synthetic sewage was measured in the presence of various concentrations of the test item after an incubation period of 3 hours.

Test Concentrations: 10, 32, 100, 320 and 1000 mg Test item/L; 1, 4 and 16 mg 3,5-Dichlorophenol/L and six inoculum controls.

 

Results

Toxicity of Test Item:

The test item was directly dosed into each test flask and pure water was added. The test item was dissolved into the pure water as homogeneously as possible.

 

Total respiration:

In comparison to the inoculum controls, the total respiration rate of the activated sludge was not inhibited at test concentrations up to 100 mg/L (inhibition below 10%) and moderately inhibited for the two highest test concentrations. The inhibition was 6.5%, -6.1% and -6.5% for the test concentrations of 10 mg/L, 32 mg/L and 100 mg/l, respectively. For the test item concentrations of 320 mg/L and 1000 mg/L, the inhibition rates were 34.4%, and 64.2%, respectively. Concentrations exceeding 1000 mg/L nominal were not tested.

The 3-hour EC10and EC50for total respiration were established to be 146.06 mg/L (95% confidence limits (cl): lower cl: 125.78 mg/L, higher cl: 169.61 mg/L) and 597.1 mg/L (95% confidence limits (cl): lower cl: 492.13 mg/L, higher cl: 718.86 mg/L), respectively; the NOEC was determined to be at a test item concentration of 100 mg/L. 

 

Respiration without nitrification (Heterotrophic Respiration):

In comparison to the inoculum controls the respiration was not inhibited for test item concentrations up to 320 mg/L and moderately inhibited at the highest test item concentration. The inhibition rates were 2.9%, 2.1%, -20.0% and -6.8% for test item concentrations of 10 mg/L, 32 mg/L, 100 mg/L and 320 mg/L, respectively. At a concentration of 1000 mg/L, the inhibition of the respiration rate was 44.4%.

The 3-hour EC10and EC50for heterotrophic respiration were established to be 842.11 mg/L (95% confidence limits (cl): lower cl: 0.00 mg/L, higher cl: n.d.) and 1012.16 mg/L (95% confidence limits (cl): lower cl: n.d., higher cl: n.d.), respectively; the NOEC was determined to be at a test item concentration of 320 mg/L. 

n.d.: not determined due to mathematical reasons

 

Respiration based on nitrification:

In comparison to the inoculum controls, the respiration rates of the activated sludge based on nitrification were almost not inhibited at test concentrations up to 100 mg/L (inhibition below 11%) and strongly inhibited for the two highest test concentrations. The inhibition was 10.5%, -15.2% and 8.5% for the test concentrations of 10 mg/L, 32 mg/L and 100 mg/l, respectively. For the test item concentrations of 320 mg/L and 1000 mg/L, the inhibition rates were 80.3%, and 86.2%, respectively. Concentrations exceeding 1000 mg/L nominal were not tested.

For the oxygen uptake due to nitrification the 3-hour EC10and EC50were established to be 99.63 mg/L (95% confidence limits (cl): lower cl: 57.75 mg/L, higher cl: 171.88 mg/L) and 201.08 mg/L (95% confidence limits (cl): lower cl: 110.45 mg/L, higher cl: 375.03 mg/L), respectively; the NOEC was determined to be at a test item concentration of 100 mg/L. 

 

This study was classified acceptable and satisfied the guideline requirements for toxicity to microorganisms studies.

Description of key information

3h-EC50 (activated sludge, total respiration inhibition) = 597.1 mg/L; 3h-EC10 (activated sludge, total respiration inhibition) = 146.06 mg/L; NOEC (activated sludge, total respiration inhibition) = 100 mg/L (nominal, OECD 209, 2016)

Key value for chemical safety assessment

EC50 for microorganisms:

597.1 mg/L

EC10 or NOEC for microorganisms:

146.06 mg/L

Additional information

In a reliable key study, toxicity of the submission substance on the activity of activated sludge was tested according to OECD 209 and in compliance with GLP (RL1). The respiration rate (oxygen consumption) of an aerobic activated sludge was measured in the presence of five test concentrations (nominal 10, 32, 100, 320 and 1000 mg Test item/L) after an incubation period of 3 hours. Six controls were run in parallel.

In comparison to the inoculum controls, the total respiration rate of the activated sludge was not inhibited at test concentrations up to 100 mg/L (inhibition below 10%) and moderately inhibited for the two highest test concentrations: For the test item concentrations of 320 mg/L and 1000 mg/L, the inhibition rates were 34.4%, and 64.2%, respectively. Derived nominal effect concentrations were: 3h-EC50 (activated sludge, total respiration inhibition) = 597.1 mg/L; 3h-EC10 (activated sludge, total respiration inhibition) = 146.06 mg/L; NOEC (activated sludge, total respiration inhibition) = 100 mg/L. Furthermore, respiration without nitrification (heterotrophic respiration) and respiration based on nitrification were measured. Derived effect concentrations were higher than effect concentrations for total respiration; therefore, relevant effect concentration for the hazard assessment were based on effect concentration based on inhibition of total respiration. 

This study was classified acceptable and satisfied the guideline requirements for toxicity to microorganisms studies. In conclusion, the test was considered reliable and adequate for the environmental hazard assessment for aquatic organisms.