2,2'-(2-methylpropylidene)bis[4,6-xylenol]

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:

15 June 2016 to 17 June 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))

Version / remarks:

Organization for Economic Co-operation and Development (OECD), OECD guidelines for Testing of Chemicals, Section 2: Effects on biotic systems, Guideline no. 209, "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation), adopted 22 July 2010.

Deviations:

yes

Remarks:

See "Any other information"

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Council regulation (EC) No 440/2008 of 30 May 2008, Part C: Methods for the determination of ecotoxicity, Publication No. L142, C 11:"Biodegradation: Activated sludge respiration inhibition test", Amended by EC No. 2016/266 of 7 December 2015, Publication No. L54.

Deviations:

yes

Remarks:

See "Any other information"

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

ISO Standard 8192, Water Quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation (2007).

Deviations:

yes

Remarks:

See "Any other information"

GLP compliance:

yes

Specific details on test material used for the study:

Test item: 207366/A
Identification: Lowinox® 22IB46
Appearance: White to cream coloured powder
Batch: WB44L0016
Purity/Composition: 99.7%
Test item storage: At room temperature
Stable under storage conditions until: 18 November 2018 (retest date)
Purity/composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Stability at higher temperatures: Not indicated
Chemical name (IUPAC), synonym or trade name: 2,2’-(2-methylpropylidene)bis[4,6-xylenol]
CAS Number: 33145-10-7
Solubility in water: Insoluble

Analytical monitoring:

not specified

Details on sampling:

Not specified

Vehicle:

no

Details on test solutions:

The batch of Lowinox® 22IB46 tested was a white to cream coloured powder with a purity of 99.7%. No correction was made for the purity/composition of the test item.
The test item was not sufficiently soluble to allow the preparation of a 10 g/L stock solution in water. Therefore, 1-Litre test bottles were filled with 200 mL of test item mixtures in Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with initial loading rates of 2.5 times the final loading rate. These mixtures were stirred in closed dark brown bottles for approximately 24 hours. Subsequently, 16 mL synthetic medium made up to 50 mL with Milli-RO water and 250 mL sludge were added resulting in the required loading rates. Optimal contact between the test item and test organisms was ensured applying continuous aeration and stirring.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Test system: Micro-organisms in activated sludge.
Source: Municipal sewage treatment plant: 'Waterschap Aa en Maas', Heeswijk Dinther, The Netherlands, receiving predominantly domestic sewage.
Preparation of the sludge: The sludge was coarsely sieved (1 mm) and allowed to settle.
The supernatant was removed and ISO-medium was added. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3 g/L of sludge, as used for the test). The pH was 7.1 on the day of testing. The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium (=sewage feed) was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
Medium: Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands)

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 temperature continuously measured in the temperature control vessels ranged between 18 and 22°C during the test, and complied with the requirements as laid down in the study plan (20 ± 2°C).

pH:

The pH in all test vessels, before addition of sludge was between 7.5 and 7.6. After the 3 hour exposure period the pH was between 7.5 and 8.1.

Dissolved oxygen:

The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the sludge flocs in suspension.

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

In a combined limit/range-finding test loading rates of 10, 100 and 1000 mg/L were tested.

Details on test conditions:

Combined limit/range-finding test
In a combined limit/range-finding test loading rates of 10, 100 and 1000 mg/L were tested.
The highest loading rate was tested in triplicate, lower loading rates consisted of one replicate. In addition, a blank control (6 replicates) and a nitrification control (2 replicates) were included. Furthermore, an abiotic control (1 replicate) and the highest loading rate with a nitrification inhibitor (3 replicates) were tested.

Contact time: 3 hours, except for the deviation described. (2 hours and 45 minutes), during which aeration and stirring took place.
Vessels: All glass open bottles/vessels.
Milli-RO water: Tap-water purified by reverse osmosis (Millipore Corp., Bedford, Mass., USA).
Synthetic medium (=sewage feed): 16 g peptone; 11 g meat extract; 3 g urea; 0.7 g NaCl; 0.4 g CaCl2.2H2O; 0.2 g MgSO4.7H2O; 2.8 g K2HPO4; Dissolved in Milli-RO water, made up to 1 litre and filtered. The pH was within 7.5 ± 0.5.
Inhibitor of nitrification: A 2.32 g/L solution of N-allylthiourea (ATU, Merck Schuchardt OHG) was prepared. 2.5 mL of this solution was added to 500 mL final test medium (final ATU concentration:
11.6 mg/L).
Air supply: Clean, oil-free air.
Aeration: The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the sludge flocs in suspension.
Performance of the test: The synthetic medium (16 mL) made up to 50 mL with Milli-RO and 200 mL test item solution were mixed (total volume 250 mL). The pH was determined. Thereafter 250 mL activated sludge was added. This was the start of the test.
After the 3-hour contact time, the oxygen consumption was recorded for a period of approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer.
The pH was determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference item concentrations and controls.
The medium temperature was recorded continuously in a temperature control vessel(s). The temperature control vessel(s) was/were identically prepared compared to the control vessels.
A temperature control vessel with a REES sensor was placed in each fume cupboard of the climate room.
Oxygen recording: Determination of oxygen was performed with multiple oxygen probes connected to a BlueBox (GO-Systemelektronik GmbH, Germany), a multichannel measuring and controlling system.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Duration:

3 h

Dose descriptor:

other: ELR50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

other: NOELR

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

Inhibition of the respiration rate
In the combined limit/range-finding test no statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 1000 mg Lowinox® 22IB46 per litre. Thus, the ELR50 was above the highest loading rate tested (1000 mg/L).
There was no significant oxygen uptake from abiotic processes and the result at 1000 mg/L with a nitrification inhibitor showed that the heterotrophic inhibition of the respiration rate was not inhibited. Based on the results of the total and heterotrophic inhibition, nitrification was calculated to be 81% inhibited. Since, the heterotrophic respiration was stimulated and nitrification is calculated using the total and heterotrophic respiration rate the inhibition of nitrification may be overestimated. Since no distorted or biphasic dose-response curve for total respiration was observed, the effect on nitrification was not required according to the guidelines and was therefore not further investigated.

Experimental conditions
The pH in all test vessels, before addition of sludge was between 7.5 and 7.6. After the 3 hour exposure period the pH was between 7.5 and 8.1.
The temperature continuously measured in the temperature control vessels ranged between 18 and 22°C during the test, and complied with the requirements as laid down in the study plan (20 ± 2°C).

Results with reference substance (positive control):

Reported in table form - see "Any other information"

Reported statistics and error estimates:

None specified

Results: pH values

Replicate	Loading rate (mg/L)	pH		Respiration rate		% Inhibition respiration rate (mean value)
		Start	End	(mg O2/L.h)	(mg O2/g.h)^1
C 1	0	7.5	7.8	45.06	30.04
C 2	0	7.5	7.8	42.59	28.39
C 3	0	7.6	7.7	56.82	37.88
C 4	0	7.6	7.8	52.79	35.19
C 5	0	7.6	7.9	42.27	28.18
C 6	0	7.6	7.7	53.85	35.90
C Mean				48.90 (RTB)	32.60
SD				6.34	4.23
CV (%)				13	13
CN 1	0	7.5	8.1	30.84	20.56
CN 2	0	7.6	8.0	38.21	25.47
CN Mean				34.53 (RHB)	23.02
R 1	2.0	7.6	8.0	33.29	22.19	32
R 2	5.0	7.6	7.9	32.88	21.92	33
R 3	12	7.6	7.9	13.99	9.33	71
T 1	10	7.6	7.9	38.48	25.65	21
T 2	100	7.6	7.8	47.48	31.65	3
T 3a	1000	7.6	7.8	47.62	31.75	3
T 3b	1000	7.6	7.8	49.40	32.93	-1
T 3c	1000	7.6	7.8	43.36	28.91	11
T 3 Mean				44.06 (RH)	29.37	-28 (IH)
TA	1000	7.6	7.5	0.5	0.33	99

C:            Blank control                                             R_TB:         Total respiration blank

CN:         Nitrification control                                      R_HB:         Heterotrophic respiration in the nitrification control

R:            Reference item, 3,5-dichlorophenol                    R_T:          Total respiration with Lowinox® 22IB46

T:            Test item, Lowinox® 22IB46                           R_H:          Heterotrophic respiration with Lowinox® 22IB46

TA:          Abiotic control of Lowinox® 22IB46                  I_T:            %inhibition of total respiration relative to R_TB

TN:         Lowinox® 22IB46 with N-allylthiourea                 I_H:           %inhibition of heterotrophic respiration relative to R_HB

SD:          Standard deviation

CV:          Coefficient of variation

¹              The amount of suspended solids in the final test mixture was 1.5 g/L.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this present test Lowinox® 22IB46 was not toxic to waste water bacteria (activated sludge) at a loading rate of 1000 mg/L (NOELR).
The ELR50 was above 1000 mg/L.

Executive summary:

The influence of Lowinox® 22IB46 on the respiration rate of activated sludge was investigated after a contact time of approximately 3 hours.

 

The study procedures described in this report were based on the OECD guideline No. 209, 2010. In addition, the procedures were designed to meet the test methods of the Council Regulation (EC) No. 440/2008 of 30 May 2008, Publication No. L142, Part C11, Amended by EC No. 2016/266 of 7 December 2015, Publication No. L54 and ISO Standard 8192 (2007).

 

The batch of Lowinox® 22IB46 tested was a white to cream coloured powder with a purity of 99.7%. No correction was made for the purity/composition of the test item.

 

The test item was not sufficiently soluble to allow the preparation of a 10 g/L stock solution in water. Therefore, the test item – Milli-RO water mixtures were magnetically stirred for a period of approximately 24 hours. Subsequently, synthetic medium, sludge and Milli-RO water were added resulting in the required loading rates. Optimal contact between the test item and test medium was ensured applying continuous aeration and stirring during the 3-hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes.

 

In a combined limit/range-finding test loading rates of 10, 100 and 1000 mg/L were tested.

The highest loading rate was tested in triplicate, lower loading rates consisted of one replicate. Furthermore, at 1000 mg/L an abiotic control (1 replicate) and three replicates with a nitrification inhibitor were tested. Responses were compared to the blank and nitrification controls.

 

No statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 1000 mg Lowinox® 22IB46 per litre.

 

The batch of activated sludge was tested for sensitivity with the reference item 3,5-dichlorophenol, and showed normal sensitivity.

 

The study met the acceptability criteria prescribed by the study plan and was considered valid.

 

Lowinox® 22IB46 was not toxic to waste water (activated sludge) bacteria at a loading rate of 1000 mg/L (NOELR).

The ELR50 was above 1000 mg/L.

Description of key information

Key value determined in a GLP accrediated laboratory study according to OECD guideline No. 209, EU Method C11 and ISO Standard 8192 (2007).

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

The influence of Lowinox® 22IB46 on the respiration rate of activated sludge was investigated after a contact time of approximately 3 hours.

 

The test item was not sufficiently soluble to allow the preparation of a 10 g/L stock solution in water. Therefore, the test item – Milli-RO water mixtures were magnetically stirred for a period of approximately 24 hours. Subsequently, synthetic medium, sludge and Milli-RO water were added resulting in the required loading rates. Optimal contact between the test item and test medium was ensured applying continuous aeration and stirring during the 3-hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes.

 

No statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 1000 mg Lowinox® 22IB46 per litre.

 

The batch of activated sludge was tested for sensitivity with the reference item 3,5-dichlorophenol, and showed normal sensitivity.

 

Lowinox® 22IB46 was not toxic to waste water (activated sludge) bacteria at a loading rate of 1000 mg/L (NOELR).

The ELR50 was above 1000 mg/L.