Fatty acids, linseed-oil, reaction products with bisphenol A-epichlorohydrin polymer and glycidyl neodecanoate

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:

From July 28, 2016 to October 06, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

data waiving: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Deviations:

yes

Remarks:

the deviations were signed and assessed by the study director and considered uncritical

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

the deviations were signed and assessed by the study director and considered uncritical

Principles of method if other than guideline:

Corresponding SOP of LAUS GmbH SOP 118 007 01 edition 10, from 15. Jan. 2016 “Belebtschlamm-Atemhemmtest“.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch no.: #210162718
Composition: Reaction products of linseed-oil fatty acids, 4,4'-methylendiphenyldiglycidylether with neodecanoic fatty acid, oxiranylmethylester
Purity: 100 % as per definition of UVCB
Appearance: brown liquid

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

In the blank control vessels, 16 mL nutrient solution was mixed with 234 mL water. The positive control vessels and the treatments were prepared by putting the appropriate amount of positive control solution respectively test substance into the respective test vessel, adding 16 mL nutrient solution and water to reach a total volume of 250 mL. Then, 250 mL inoculum was added in 5 minute intervals and the mixtures were aerated. After 3 hours, the content of the first vessel was poured in a 250 mL narrow-neck bottle and the respiration rate was determined by measurement of the O2-concentration over a period of max. 5 minutes. The following vessels were measured likewise in 5 minute intervals.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Activated sludge from a biologic sewage treatment plant was used. The chosen plant treats mostly domestic sewage. The sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant in D-67435 NW-Lachen-Speyerdorf. Upon arrival in the test facility, the sludge was filtrated, washed with tap water 3 times and re-suspended in tap water. The activated sludge was aerated until usage in the test and fed daily with 50 mL synthetic sewage feed /L.
First experiment: the dry matter was determined as 2.76 g suspended solids/L, giving a concentration of 1.38 g suspended solids/L in the test.
Second experiment: the dry matter of the inoculum was determined as 2.98 g suspended solids/L, giving a concentration of 1.49 g suspended solids/L in the test.

Test type:

static

Water media type:

freshwater

Total exposure duration:

3 h

Hardness:

1.03 mmol/L

Test temperature:

19.3 - 22.2°C

pH:

8.05

Salinity:

-

Conductivity:

242 µS/cm

Nominal and measured concentrations:

0, 1, 3.2, 10, 32, 100, 320 and 1000 mg/L (nominal)

Details on test conditions:

Glass beakers (1000 ml) were used as test vessels. Narrow-neck glass bottles with flat bottoms (250 mL) were used as measuring flasks.
Duration: 3 hours
Replicates: 1 replicate/treatment (positive control, all experiments), 1 replicate/concentration test substance 100, 10 and 1 mg/L (first experiment), 5 replicates concentration test substance 1000 mg/L (first experiment) and 5 replicates test substance treatment 320, 100, 32, 10 and 3.2 mg/L (second experiment)
Blank control: 2 replicates before and 2 after measuring positive control and test substance, respectively
Water: tap water
Aeration: purified air, using Pasteur pipettes
Feeding: nutrient solution, 16 mL/vessel

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol (CAS-No. 591-35-5)

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

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

- In the first experiment (a range finder pre-test), the test substance was tested using 4 nominal concentrations ranging from 1000 to 1 mg/L. At the beginning of oxygen measurement, the oxygen concentration in the test solutions was much lower than in the positive control and the control solutions. The low oxygen concentration in the test solution was very likely caused by the poor solubility of the test substance. Indeed, a thin layer of test substance was observed on the surface of the test solutions, resulting in some bias of the gas exchange between liquid phase and atmosphere. Therefore, the duration for oxygen measurement in the test solutions was strongly reduced (to less than 5 min). (Because linearity of all regression curves was given, this could be stated as uncritical). As a result, the inhibition values in the test solutions were in a similar range for all concentrations and strongly scattered. At the highest concentrated treatment of 1000 mg/L, significant inhibition was observed. This inhibition at 1000 mg/L did however not rise above 23.2 % and therefore the EC50 could be stated as > 1000 mg/L. Because of the scattering inhibition values no graphic determination of the EC10 was possible.
- In order to determine a NOEC value, a second experiment (main test) was performed under the same conditions. In this experiment, 5 concentrations of the test substance ranging from 320 to 3.2 mg/L were used. No inhibition was observed and oxygen concentration in the test solution was in the same range as in the control. The following results for the test substance were determined: a 3h NOEC of 320 mg/L and a 3h EC50 of >1000 mg/L
- All validity criteria were met. For the estimation of the EC50 of the positive control, the fits showed good statistical correspondence of the data with the dose-response-equation. The positive control gave an EC50 of 9.2 mg/L (first experiment) and 14 mg/L (second experiment) which lie within the range of 2 – 25 mg/L. The coefficient of variation of oxygen uptake rate in control replicates was below 30% at the end of the test. The oxygen uptake rate of the blank controls was above 20 mg O2 per gram activated sludge in 1 hour. No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test can be considered valid.

Results with reference substance (positive control):

3,5-dichlorophenol was used as positive control. Four concentrations were tested.
First experiment: an EC50 of 9.2 mg/L (95% confidence interval: 4.7 – 13 mg/L) was determined, which lies within the range of 2 – 25 mg/L (OECD guideline).
Second experiment: an EC50 of 14 mg/L (95% confidence interval: 7.9 – 24 mg/L) was determined. The value lies within the range of 2 – 25 mg/L (OECD guideline).

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 3 h NOEC and EC50 values for respiration inhibition of activated sludge due to the test substance were determined to be at 320 and >1000 mg/L (nominal) respectively.

Executive summary:

A study was conducted to determine the toxicity of the test substance to microorganisms according to OECD Guideline 209 and EU Method C.11 (respiration inhibition test), in compliance with GLP. Activated sludge was exposed to the test substance at nominal concentrations of 0, 10, 32, 100, 320, and 1000 mg/L for 3 h. In addition, two controls and four different concentrations of the reference substance 3,5-dichlorophenol were tested. The respiration rate (oxygen consumption) of the aerobic activated sludge fed with a standard amount of synthetic wastewater was measured. The inhibitory effect of the test substance at the particular concentrations was expressed as a percentage of the mean respiration rate of the controls. Two experiments were performed. In the first experiment (a range finder pre-test), the test substance was tested using 4 nominal concentrations ranging from 1000 to 1 mg/L. At the beginning of oxygen measurement, the oxygen concentration in the test solutions was much lower than in the positive control and the control solutions. The low oxygen concentration in the test solution was very likely caused by the poor solubility of the test substance. Indeed, a thin layer of test substance was observed on the surface of the test solutions, resulting in some bias of the gas exchange between liquid phase and atmosphere. Therefore, the duration for oxygen measurement in the test solutions was strongly reduced (to less than 5 min). (Because linearity of all regression curves was given, this could be stated as uncritical). As a result, the inhibition values in the test solutions were in a similar range for all concentrations and strongly scattered. At the highest concentrated treatment of 1000 mg/L, significant inhibition was observed. This inhibition at 1000 mg/L did however not rise above 23.2 % and therefore the EC50 could be stated as > 1000 mg/L. Because of the scattering inhibition values no graphic determination of the EC10 was possible. In order to determine a NOEC value, a second experiment (main test) was performed under the same conditions. In this experiment, 5 concentrations of the test substance ranging from 320 to 3.2 mg/L were used. No inhibition was observed and oxygen concentration in the test solution was in the same range as in the control. The following results for the test substance were determined: a 3 h NOEC of 320 mg/L and a 3h EC50 of >1000 mg/L. All validity criteria were met. For the estimation of the EC50 of the positive control, the fits showed good statistical correspondence of the data with the dose-response-equation. The positive control gave an EC50 of 9.2 mg/L (first experiment) and 14 mg/L (second experiment) which lie within the range of 2 – 25 mg/L. The coefficient of variation of oxygen uptake rate in control replicates was below 30% at the end of the test. The oxygen uptake rate of the blank controls was above 20 mg O2 per gram activated sludge in 1 h. No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test could be considered valid. Under the study conditions, the 3 h NOEC and EC50 values for respiration inhibition of activated sludge due to the test substance were determined to be at 320 and >1000 mg/L (nominal) respectively (Muckle, 2016).

Description of key information

The 3 h NOEC and EC50 values for respiration inhibition of activated sludge due to the test substance were determined to be at 320 and >1000 mg/L (nominal) respectively.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

320 mg/L

Additional information

A study was conducted to determine the toxicity of the test substance to microorganisms according to OECD Guideline 209 and EU Method C.11 (respiration inhibition test), in compliance with GLP. Activated sludge was exposed to the test substance at nominal concentrations of 0, 10, 32, 100, 320, and 1000 mg/L for 3 h. In addition, two controls and four different concentrations of the reference substance 3,5-dichlorophenol were tested. The respiration rate (oxygen consumption) of the aerobic activated sludge fed with a standard amount of synthetic wastewater was measured. The inhibitory effect of the test substance at the particular concentrations was expressed as a percentage of the mean respiration rate of the controls. Two experiments were performed. In the first experiment (a range finder pre-test), the test substance was tested using 4 nominal concentrations ranging from 1000 to 1 mg/L. At the beginning of oxygen measurement, the oxygen concentration in the test solutions was much lower than in the positive control and the control solutions. The low oxygen concentration in the test solution was very likely caused by the poor solubility of the test substance. Indeed, a thin layer of test substance was observed on the surface of the test solutions, resulting in some bias of the gas exchange between liquid phase and atmosphere. Therefore, the duration for oxygen measurement in the test solutions was strongly reduced (to less than 5 min). (Because linearity of all regression curves was given, this could be stated as uncritical). As a result, the inhibition values in the test solutions were in a similar range for all concentrations and strongly scattered. At the highest concentrated treatment of 1000 mg/L, significant inhibition was observed. This inhibition at 1000 mg/L did however not rise above 23.2 % and therefore the EC50 could be stated as > 1000 mg/L. Because of the scattering inhibition values no graphic determination of the EC10 was possible. In order to determine a NOEC value, a second experiment (main test) was performed under the same conditions. In this experiment, 5 concentrations of the test substance ranging from 320 to 3.2 mg/L were used. No inhibition was observed and oxygen concentration in the test solution was in the same range as in the control. The following results for the test substance were determined: a 3 h NOEC of 320 mg/L and a 3h EC50 of >1000 mg/L. All validity criteria were met. For the estimation of the EC50 of the positive control, the fits showed good statistical correspondence of the data with the dose-response-equation. The positive control gave an EC50 of 9.2 mg/L (first experiment) and 14 mg/L (second experiment) which lie within the range of 2 – 25 mg/L. The coefficient of variation of oxygen uptake rate in control replicates was below 30% at the end of the test. The oxygen uptake rate of the blank controls was above 20 mg O2 per gram activated sludge in 1 h. No observations were made which might cause doubts concerning the validity of the study outcome. The result of the test could be considered valid. Under the study conditions, the 3 h NOEC and EC50 values for respiration inhibition of activated sludge due to the test substance were determined to be at 320 and >1000 mg/L (nominal) respectively (Muckle, 2016).