Hexamethylene diisocyanate, oligomers, reaction products with 3-methoxypropylamine

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 January 18, 2017 to January 19, 2017

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:

yes

Remarks:

see 'Principles of method if other than guideline'

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

see 'Principles of method if other than guideline'

Principles of method if other than guideline:

The following deviation from the study plan was recorded:
- The measurement period was partly less than 5 minutes. Because linearity of all regression curves was given and the correlation of the inhibition values among the test
replicates was good, this was stated as uncritical.
The deviation was assessed and signed by the study director on 25. Jan. 2017.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

CAS No.: 162492-07-1; EINECS-No. 500-740-9; Batch No.: 1591ZG-101; Purity >98%; Appearance: white powder

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Due to the poor solubility of the test substance, the test substance was directly weighed into the test vessels. For best possible bioavailability of the poorly soluble test substance, a homogenous dispersion of the test substance in tap water was prepared. For this the respective loading rate in 234 mL water was stirred on a magnetic stirrer for 25 hours. The resulting solution was used for preparation of the treatments.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Source:
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.

Pre-treatment:
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.
One day before start of the test, the inoculum was taken from its source, washed, aerated and the dry matter was determined. Volume was adapted to the desired content of dry matter. The nutrient solution was thawed and the sludge was fed with 50 mL nutrient solution / L sludge. On the day of the experiment, the dry matter was determined once more. The stock solution of the positive control was prepared.
On the next day, in the blank control vessels, 16 mL nutrient solution was mixed with 234 mL water. The positive control vessels were prepared by putting the appropriate amount of positive control solution into the respective test vessel, adding 16 mL nutrient solution and water to reach a total volume of 250 mL. In the treatments 16 mL nutrient solution was added to the pre-treated solutions in order to obtain a total volume of 250 mL. Then, 250 mL inoculum was added in 5 minutes 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.


Dry matter of Sludge (g suspended solids/L): 3.20
Dry matter in the Test (g suspended solids/L): 1.60

Test type:

static

Water media type:

other: tap water

Limit test:

no

Total exposure duration:

3 h

Test temperature:

19.3 – 20.9 °C

pH:

7.7

Nominal and measured concentrations:

Nominal, from 1000 to 1 mg/L.

Details on test conditions:

Duration: 3 hours
Replicates: 1 replicate/concentration (positive control, test substance lower concentrations); 5 replicates/test substance treatment 1000 mg/L
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

Replicates:
The test substance was tested using 4 concentrations ranging from 1000 to 1 mg/L nominal concentration. For the treatment 1000 mg/L, 5 replicates were used; for the other treatments, 1 replicate each.

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

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

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

No significant inhibition was observed caused by the treatment with the test substance.
The difference between treatment 1000 mg/L and the blank control can be considered as not significant, because O2 consumption in the treatment was higher than in the blank control. Therefore, the concentration 1000 mg/L is stated as NOEC.

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.

Results with reference substance (positive control):

3,5-Dichlorophenol was used as positive control. Four concentrations were tested; an EC50 of 7.5 mg/L (95% confidence interval: 4.8 - 10 mg/L) was determined, which lies within the recommended range of the OECD guideline (2 – 25 mg/L).

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the NOEC ≥ 1000 mg/L, 3h EC50 was >1000 mg/L (nominal). The 3h EC10 and NOEC were both 1000 mg/L.

Executive summary:

A study was conducted to determine the effect of the test substance on the respiration of activated sludge obtained from a domestic sewage treatment plant according to OECD Guideline 209 and EU Method C.11, in compliance with GLP. The activated sludge was taken from a domestic sewage treatment plant and washed before usage. The dry matter of the inoculum was determined as 3.2 g suspended solids/L, giving a concentration of 1.6 g suspended solids/L in the assay. Due to the poor solubility, the test substance was weighed directly into the vessels where later sludge was added. One day before start of the experiment, the substance was weighed into the test vessels, mixed with 234 mL tap water and stirred for 24 h on magnetic stirrers. The substance was tested using 4 concentrations ranging from 1000 to 1 mg/L (nominal). Five replicates were used for the treatment with the highest tested concentration 1000 mg/L and 1 replicate for the remaining treatments. 3,5-Dichlorophenol was the positive control, with an EC50 of 7.5 mg/L lying within the recommended range of the OECD guideline (2 – 25 mg/L). All other validity criteria of the test were also met. Because no significant inhibition was observed, no additional experiment was performed. Under the study conditions, the NOEC ≥ 1000 mg/L, 3h EC50 was >1000 mg/L (nominal). The 3h EC10 and NOEC were both 1000 mg/L (nominal) (Muckle, 2017).

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

A study was conducted to determine the effect of the test substance on the respiration of activated sludge obtained from a domestic sewage treatment plant according to OECD Guideline 209 and EU Method C.11, in compliance with GLP. The activated sludge was taken from a domestic sewage treatment plant and washed before usage. The dry matter of the inoculum was determined as 3.2 g suspended solids/L, giving a concentration of 1.6 g suspended solids/L in the assay. Due to the poor solubility, the test substance was weighed directly into the vessels where later sludge was added. One day before start of the experiment, the substance was weighed into the test vessels, mixed with 234 mL tap water and stirred for 24 h on magnetic stirrers. The substance was tested using 4 concentrations ranging from 1000 to 1 mg/L (nominal). Five replicates were used for the treatment with the highest tested concentration 1000 mg/L and 1 replicate for the remaining treatments. 3,5-Dichlorophenol was the positive control, with an EC50 of 7.5 mg/L lying within the recommended range of the OECD guideline (2 – 25 mg/L). All other validity criteria of the test were also met. Because no significant inhibition was observed, no additional experiment was performed. Under the study conditions, the NOEC ≥ 1000 mg/L, 3h EC50 was >1000 mg/L (nominal). The 3h EC10 and NOEC were both 1000 mg/L (nominal) (Muckle, 2017).