Reaction mass of 2-ethylhexyl (3-isocyanato-4-methylphenyl)carbamate and 2-ethylhexyl (5-isocyanato-2-methylphenyl)carbamate and 2-ethylhexyl (3-isocyanato-2-methylphenyl)carbamate

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:

May 15, 2018 to July 9, 2018

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))

Version / remarks:

Organisation for Economic Cooperation and Development. 2010. Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation). OECD Guideline for Testing of Chemicals, Guideline 209. Adopted 22 July 2010.

Deviations:

not specified

GLP compliance:

yes

Specific details on test material used for the study:

No further details specified in the study report.

Analytical monitoring:

not specified

Vehicle:

no

Details on test solutions:

Test mixtures were prepared at 15 minute intervals starting with the first control. Each control vessel contained 16.0 mL of synthetic sewage, 234 mL of deionized water, and 250 mL of inoculum for a total volume of 500 mL. The mixture was promptly aerated at a rate sufficient to keep the solids in suspension. Subsequent reference and test substance dosed biotic mixtures were prepared by combining 16.0 mL of synthetic sewage, the appropriate volume of reference substance stock solution or appropriate amount of test substance and an appropriate volume of deionized water needed to achieve total volume of 250 mL.
After the addition of deionized water, 250 mL of inoculum was added into each test vessel to bring the total volume to 500 mL and the vessel was placed under aeration for three hours prior to start DO measurements.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Activated sludge collected from the Easton Wastewater Treatment Facility, Easton, Maryland was used to prepare inoculum for the test. The Easton facility collects waste waters predominately from domestic sources. The sludge was sieved using a 2 mm screen and was kept under aeration in temperature controlled room. Fifty mL of synthetic sewage per liter of the sludge was added at the end of each day.
Prior to use, the sludge was allowed to settle for 30 minutes and the supernatant above the settled solids was removed. Total suspended solids (TSS) concentration of the sludge was determined. The inoculum was prepared by diluting of an aliquot of the settled sludge with an aliquot of deionized (reverse osmosis) water to achieve target TSS concentration of ~3000 mg/L. The inoculum was maintained at an average temperature of 20 ± 2 °C, continuously aerated, and used for testing the same day. The pH and TSS concentration of the inoculum were determined prior to use on the day of the study.

Test type:

flow-through

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

No post exposure observation period specified.

Hardness:

Not specified

Test temperature:

The temperatures in the temperature controlled room during the test ranged from 19.5 to 20.1 °C.

pH:

The results of the pre-test pH evaluations indicated that after addition of the test substance, the pH of the inoculum/synthetic sewage/water mixture remained within 7.5±0.5 range.

Dissolved oxygen:

Not applicable

Salinity:

Not applicable

Conductivity:

Not specified

Nominal and measured concentrations:

Nominal concentrations: 10, 100 & 1000 mg/L

Details on test conditions:

Test Conditions and Apparatus
Control, reference, and treatment test mixtures were incubated at 20 ± 2ºC and aerated for three hours at a rate sufficient to provide aerobic conditions and maintain solids in suspension. The mixtures were prepared and aerated in 1000-mL Erlenmeyer flasks and then transferred to fill 300-mL biochemical oxygen demand (BOD) bottles to conduct dissolved oxygen (DO) measurements. Each test flask was identified by project number, test substance identification, test concentration, and a flask number.

Pre-test pH Evaluation
Effect of the test substance on the pH of the inoculum/water mixtures was assessed prior to performing of the test. A pH pre-test evaluation of the test substance treated mixtures was performed using test mixtures prepared by placing 16 mL of synthetic sewage, 250 mL of sludge inoculum, the appropriate amount of deionized water to bring the total volume to 500 mL , and the appropriate amount of test substance into a 1-L Erlenmeyer flask.

Sample Analysis
After 3 hours of aeration, the contents of the first control vessel was transferred to a BOD bottle and dissolved oxygen concentration was measured every 10 seconds over a 10 minute period or until the DO measurement dropped below 2.0 mg O2/L using a YSI Model 5000 Dissolved Oxygen Meter. The respiration rate in all other vessels was determined in an identical manner at 15 minute intervals. The contact time for each concentration of reference or test substance with the inoculum was three hours.

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

Details on results:

The respiration rates in biotic test mixtures dosed with the test substance at concentrations of 10 and 100 mg/L were 32.4 and 33.0 mg O2/L/h, respectively. Compared to the untreated control test mixtures, respiration inhibition of the test substance treated test vessels at concentrations of 10 and 100 mg /L were -4.0% and -5.9%, respectively. Average (of 3 replicates) respiration rate in biotic test mixture dosed with the test substance at 1000 mg/L was 26.5 mg O2/L/h (Table 3) and average respiration inhibition was 15%.
The abiotic treatment mixture dosed with 1000 mg/L of the test substance provided a respiration rate of 0.5 mg O2/L/h indicating that there was some uptake of oxygen resulting from abiotic reactions of the test substance. Average respiration inhibition in biotic test mixture dosed with the test substance at 1000 mg/L adjusted by the value of abiotic respiration was 16.6%.

Results with reference substance (positive control):

The validity of the test was also supported by the data for the 3,5-dichlorophenol reference group, which resulted in an EC50 value of 8.6 mg/L, with 95% confidence limits of 6.8 mg/L and 11.5 mg/L. This EC50 value was within the 2 to 25 mg/L range considered acceptable for the test.

 Pre-Test pH Evaluation

Vessel ID	Trixene AS concentration, mg/L	Measured pH
pH Mixture 1 pH Mixture 2 pH Mixture 3 pH Mixture 4	0 10 100 1000	7.37 7.41 7.45 7.51

 

Dissolved Oxygen (DO) Concentration in the Test Mixtures

Time, s	Dissolved oxygen concentrations, mg O2/L
	Control 1	Control 2	2,5-dichlorophenol, mg/L			Trixene AS, mg/L
			3	15	50	10	100	1000	1000	1000	1000
								Rep. 1	Rep. 2	Rep. 3	Abiotic
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 500 510 520 530 540 550 560 570 580 590 600	7.47 6.80 6.61 6.53 6.45 6.35 6.27 6.19 6.10 6.03 5.96 5.86 5.78 5.71 5.61 5.54 5.47 5.39 5.30 5.21 5.11 5.06 4.97 4.90 4.82 4.75 4.66 4.58 4.51 4.44 4.37 4.28 4.20 4.11 4.02 3.96 3.87 3.80 3.71 3.63 3.56 3.48 3.41 3.32 3.25 3.17 3.08 2.99 2.93 2.84 2.77 2.70 2.62 2.53 2.46 2.37 2.29 2.22 2.13 2.06	9.01 6.41 5.77 5.57 5.47 5.37 5.29 5.19 5.08 4.99 4.89 4.79 4.70 4.60 4.51 4.42 4.34 4.23 5.15 4.06 3.96 3.86 3.78 3.68 3.59 3.50 3.42 3.31 3.22 3.14 3.03 2.93 2.86 2.76 2.67 2.58 2.49 2.39 2.31 2.22 2.11 2.03 1.93	7.17 6.44 6.29 6.22 6.14 6.07 5.99 5.92 5.84 5.75 5.71 5.62 5.56 5.47 5.41 5.35 5.29 5.23 5.15 5.09 5.03 4.94 4.87 4.80 4.73 4.68 4.61 4.55 4.47 4.41 4.33 4.26 4.20 4.13 4.08 4.01 3.93 3.87 3.81 3.74 3.66 3.60 3.52 3.46 3.40 3.33 3.25 3.19 3.11 3.05 2.99 2.91 2.85 2.79 2.73 2.66 2.59 2.53 2.45 2.38	7.95 7.09 6.98 6.92 6.88 6.85 6.81 6.77 6.75 6.71 6.67 6.64 6.61 6.58 6.55 6.52 6.49 6.45 6.43 6.40 6.38 6.35 6.31 6.29 6.26 6.23 6.20 6.17 6.14 6.11 6.09 6.05 6.02 6.00 5.97 5.94 5.92 5.89 5.86 5.83 5.81 5.77 5.75 5.72 5.69 5.66 5.63 5.60 5.58 5.54 5.52 5.49 5.46 5.43 5.41 5.38 5.35 5.32 5.30 5.27	9.04 8.30 8.17 8.14 8.12 8.09 8.08 8.07 8.05 8.03 8.02 8.01 7.99 7.99 7.98 7.97 7.96 7.95 7.94 7.93 7.92 7.92 7.91 7.90 7.89 7.88 7.87 7.86 7.85 7.84 7.83 7.82 7.81 7.80 7.79 7.78 7.78 7.77 7.76 7.75 7.74 7.73 7.72 7.71 7.70 7.69 7.68 7.68 7.67 7.66 7.65 7.65 7.64 7.63 7.61 7.60 7.59 7.58 7.57 7.56	8.48 2.98 2.44 2.30 2.21 2.12 2.02 1.94	8.88 4.72 4.28 4.14 4.04 3.93 3.84 3.75 3.66 3.55 3.47 3.38 3.28 3.19 3.11 3.01 2.93 2.83 2.74 2.66 2.57 2.47 2.39 2.28 2.20 2.12 2.04 1.94	9.02 6.25 5.21 5.01 4.91 4.79 4.71 4.61 4.52 4.44 4.36 4.26 4.19 4.11 4.01 3.93 3.84 3.76 3.68 3.61 3.53 3.44 3.36 3.27 3.19 3.11 3.04 2.94 2.87 2.77 2.70 2.63 2.55 2.46 2.38 2.31 2.21 2.12 2.06 1.97	8.80 5.92 5.45 5.25 5.16 5.06 4.98 4.90 4.81 4.72 4.67 4.59 4.49 4.43 4.35 4.26 4.19 4.12 4.03 3.95 3.89 3.80 3.74 3.66 3.58 3.51 3.43 3.34 3.28 3.21 3.12 3.05 2.98 2.91 2.82 2.75 2.68 2.60 2.52 2.44 2.37 2.30 2.23 2.14 2.07 1.99	8.97 5.95 5.26 5.16 5.08 5.02 4.94 4.88 4.81 4.76 4.69 4.64 4.59 4.52 4.46 4.41 4.34 4.28 4.23 4.17 4.11 4.06 3.99 3.93 3.87 3.80 3.76 3.71 3.64 3.58 3.53 3.48 3.41 3.36 3.24 3.18 3.13 3.08 3.01 2.96 2.89 2.84 2.79 2.73 2.67 2.60 2.55 2.50 2.43 2.38 2.32 2.27 2.22 2.15 2.10 2.05 1.98	9.15 8.88 8.42 8.33 8.30 8.28 8.27 8.26 8.26 8.26 8.25 8.25 8.24 8.24 8.24 8.24 8.24 8.24 8.24 8.24 8.24 8.24 8.24 8.24 8.24 8.24 8.24 8.24 8.24 8.24 8.24 8.24 8.24 8.24 8.24 8.24 8.24 8.24 8.24 8.24 8.24 8.24 8.24 8.24 8.24 8.24 8.24 8.24 8.24 8.24 8.24 8.24 8.24 8.25 8.25 8.25 8.25 8.25 8.25 8.25

Range of dissolved oxygen values used to calculated respiration rates are shown in bold.

 

Respiration Rates and Percent Inhibition

Treatment	Nominal concentration, mg/L	Respiration rate, mg O2/L/h^1	Average respiration rate, mg O2/L/h^1	Respiration inhibition, %^1	Average respiration inhibition, %^1
Control 1 Control 2	0 0	28.7 33.6	31.2	NA NA
3,5-dichlorophenol 3,5-dichlorophenol 3,5-dichlorophenol	3 15 50	24.7 10.6 3.7		20.8 66.0 88.0
Trixene AS Trixene AS	10 100	32.4 33.0		-4.0 -5.9
Trixene AS       Rep. 1 Trixene AS       Rep. 2 Trixene AS       Rep. 3	1000 1000 1000	30.4 27.9 21.2	26.5	2.5 10.3 32.0	15
Trixene AS Abiotic	1000	0.5		NA
^1Calculations were performed in Excel in full precision mode. Calculations based on rounded numbers presented in the tables may provide slightly different results.

 

Acceptance Criteria of the Test

Parameter	Test value	Units	Acceptance criteria
Oxygen uptake in Controls per hour Average oxygen uptake of Control 1 and Control 2 Coefficient of Variation of oxygen uptake in Controls	3.12 11.0	mg O2/L/h %	≤30%
Oxygen uptake in Controls per g of activated sludge solids            Control 1            Control 2            Average	18.9 22.1 20.5	mg O2/L/h mg O2/L/h mg O2/L/h	≥20 mg O2/L/h
EC50 in Reference Controls            EC50 (3 hours) of 3,5-dichlorophenol	8.6	mg/L	2 to 25 mg/L

 

Validity criteria fulfilled:

yes

Conclusions:

Under the test conditions, Trixene AS did not provide activated sludge respiration inhibition at the dose levels of 10 and 100 mg/L. At the highest tested dose level of 1000 mg/L respiration inhibition (average of three replicates) was 15%. The abiotic treatment mixture dosed with 1000 mg/L of Trixene AS provided respiration rate of 0.5 mg O2/L/hr showing that there was no significant uptake or release of oxygen resulting from abiotic reactions of the test substance. The EC50 value for Trixene AS is greater than 1000 mg/L, the highest concentration tested.

Executive summary:

The study was performed to assess the effects of the test substance on activated sludge microorganisms by determining the respiration rate at the EAG Laboratories environmental fate facility in Easton, Maryland. This study was performed based on procedures in the OECD Guideline for the Testing of Chemicals, Guideline 209: Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation).

 

The test contained control, reference, and treatment groups. The control replicates were used to determine the background respiration rate of the sludge and were not exposed to the test or reference substances. The reference group was dosed with 3, 5-dichlorophenol, a known inhibitor of respiration, at concentrations of 3, 15, and 50 mg/L. The test substance was tested at nominal dose levels of 10, 100, and 1000 mg/L. The 1000 mg/L treatment was tested in triplicate. An abiotic control was treated with the test substance at a dose level of 1000 mg/L to examine the potential for abiotic reactions of the test substance to consume or release oxygen under the tested conditions.

 

Under the test conditions, Trixene AS did not provide activated sludge respiration inhibition at the dose levels of 10 and 100 mg/L. At the highest tested dose level of 1000 mg/L respiration inhibition (average of three replicates) was 15%. The abiotic treatment mixture dosed with 1000 mg/L of Trixene AS provided respiration rate of 0.5 mg O2/L/hr showing that there was no significant uptake or release of oxygen resulting from abiotic reactions of the test substance. The EC50 value for Trixene AS is greater than 1000 mg/L, the highest concentration tested.

Description of key information

The EC50 value for Trixene AS is greater than 1000 mg/L, the highest concentration tested.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

Additional information

The study was performed to assess the effects of the test substance on activated sludge microorganisms by determining the respiration rate. This study was performed based on procedures in the OECD Guideline for the Testing of Chemicals, Guideline 209: Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation).

The test contained control, reference, and treatment groups. The reference group was dosed with 3, 5-dichlorophenol, a known inhibitor of respiration, at concentrations of 3, 15, and 50 mg/L. The test substance was tested at nominal dose levels of 10, 100, and 1000 mg/L. The 1000 mg/L treatment was tested in triplicate. An abiotic control was treated with the test substance at a dose level of 1000 mg/L to examine the potential for abiotic reactions of the test substance to consume or release oxygen under the tested conditions.

Under the test conditions, Trixene AS did not provide activated sludge respiration inhibition at the dose levels of 10 and 100 mg/L. At the highest tested dose level of 1000 mg/L respiration inhibition (average of three replicates) was 15%. The abiotic treatment mixture dosed with 1000 mg/L of Trixene AS provided respiration rate of 0.5 mg O2/L/hr showing that there was no significant uptake or release of oxygen resulting from abiotic reactions of the test substance. The EC50 value for Trixene AS is greater than 1000 mg/L, the highest concentration tested.