4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with [(dimethylamino)methyl]phenol and piperazine

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:

This study was conducted between 11 September 2017 and 12 September 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))

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Information as provided by the Sponsor.
Identification: THEMIS (EC 500-429-8)
Physical state/Appearance: pale brown powder
CAS Number: 159034-96-5
EC Number: EC 500-429-8
Batch: 161129
Purity: 100%
Expiry Date: 28 November 2017

Analytical monitoring:

no

Remarks:

As it was not a requirement of the Test Guidelines, no analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and is reflected in the GLP compliance statement.

Vehicle:

no

Details on test solutions:

Range-Finding Test
Test Item Preparation
In the range-finding test, activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/L. The test item was dispersed directly in water.
Nominal amounts of test item (5, 50 and 500 mg (500 mg in triplicate)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours, at room temperature, in order to maximize the dissolved test item concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 100 and 1000 mg/L (3 replicates).
The control group was maintained under identical conditions but not exposed to the test item.

Reference Item Preparation
A reference item, 3,5-dichlorophenol, was included in the range-finding test at concentrations of 3.2, 10 and 32 mg/L in order to confirm the suitability of the inoculum. A stock solution of 0.5 g/L was prepared by dissolving the reference item directly in water with the aid of ultrasonication for approximately 15 minutes. The pH of this stock solution was measured to be pH 6.1 and was adjusted to pH 7.1 using 1.0 M NaOH. The pH values were measured using a Hach HQ40d Flexi handheld meter. Aliquots (3.2, 10 and 32 mL) of the stock solution were removed and dispersed with activated sewage sludge (250 mL), synthetic sewage (16 mL) and water to a final volume of 500 mL to give the required concentrations of 3.2, 10 and 32 mg/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

A mixed population of activated sewage sludge micro-organisms was obtained on 11 September 2017 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.

Preparation of Inoculum
The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC overnight prior to use in the test. On the day of collection the activated sewage sludge (6 liters) was fed synthetic sewage (300 mL). The pH of the sample on the day of the test was 7.3 measured using a Hach HQ40d Flexi handheld meter. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper using a Buchner funnel which was then rinsed 3 times with 10 mL of deionized reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105 ºC for at least one hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.0 g/L prior to use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

Not reported

Test temperature:

19 -20”C

pH:

7.8-7.9

Dissolved oxygen:

7.85 - 8.75 mg O2/L or 88 - 98% of Dissolved Oxygen Saturation Level of 8.9 mg O2/L

Salinity:

Not reported

Conductivity:

Not reported

Nominal and measured concentrations:

Range-Finding Test
Nominal test concentrations of 10, 100 and 1000 mg/L. The test item was dispersed directly in water.

Test Item Preparation
Nominal amounts of test item (5, 50 and 500 mg (500 mg in triplicate)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours, at room temperature, in order to maximize the dissolved test item concentration. All test vessels were shielded from the light during mixing. Synthetic sewage (16 mL), activated sewage sludge (250 mL) and water were added to a final volume of 500 mL to give the required concentrations of 10, 100 and 1000 mg/L (3 replicates).

Details on test conditions:

Preparation of Test System
At time "0" 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum added in a 500 mL conical flask (first control). The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of 0.5 to 1.0 liter per minute. Thereafter, at 15 minute intervals the procedure was repeated for the second control followed by the reference item vessels with appropriate amounts of the reference item being added. The test item vessels were prepared as described above above under "Details of Test Solutions". Finally two further control vessels were prepared.

The test was conducted under normal laboratory lighting in a temperature controlled room at measured temperatures of between 19 and 20 ºC.

Reference substance (positive control):

yes

Remarks:

Information as provided by the Supplier (Sigma-Aldrich). Identification: 3,5-Dichlorophenol Physical state/Appearance: pale brown crystalline solid Batch: MKBZ0947V Purity: 97.0% Expiry Date: 01 August 2022 Storage Conditions: room temperature

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

Details on results:

Range-Finding Test
The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60% of the dissolved oxygen saturation level of 8.9 mg O2/L.
No statistically significant toxic effects were shown at any of the test concentrations employed. It was therefore considered justifiable not to perform a definitive test.
It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L

Validation Criteria
The coefficient of variation of oxygen uptake in the control vessels was 3.0% and the specific respiration rate of the controls was 25.40 mg oxygen per gram dry weight of sludge per hour. The validation criteria have therefore been satisfied.
The validation criterion for the reference item EC50 value was also satisfied .

Inhibition of Respiration Rate
The following results were derived:
Test Item 3,5-dichlorophenol
ECx (3 Hours) (mg/L) 95% Confidence Limits (mg/L) ECx (3 Hours) (mg/L) 95% Confidence Limits (mg/L)
EC10 <1000 - 0.74 -
EC20 <1000 - 1.2 -
EC50 >1000 4.8 3.5 - 6.6
EC80 >1000 - 20 -
NOEC 1000 - - -

In some instances, the initial and final dissolved oxygen concentrations were outside those recommended in the test guidelines (7 mg O2/L and 2 mg O2/L respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.

Results with reference substance (positive control):

See "Details on Results" (above)

Table 1: Dissolved Oxygen Concentrations of the Test Preparations after 30 Minutes Contact Time in the Range-Finding Test

Nominal Concentration (mg/L)		Dissolved Oxygen Concentration (mg O2/L)	Expressed as a Percentage of a Dissolved Oxygen Saturation Level of 8.9 mg O2/L
Control	R1	8.36	94
	R2	8.45	95
	R3	8.22	92
	R4	8.26	93
Test Item	10	7.92	89
	100	8.45	95
	1000 R1	7.85	88
	1000 R2	8.33	94
	1000 R3	8.20	92
3,5-dichlorophenol	3.2	8.75	98
	10	7.90	89
	32	8.55	96

R₁– R₄= Replicates 1 to 4

Table 2: Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time in the Range-Finding Test

Nominal Concentration (mg/L)		Initial O2Reading (mg O2/L)	Measurement Period (minutes)	Final O2 Reading (mg O2/L)	O2 Consumption Rates (mg O2/L/hour)	% Inhibition
Control	R1	6.0	5	2.7	39.60	-
	R2	5.7	5	2.6	37.20	-
	R3	5.4	5	2.2	38.40	-
	R4	5.7	5	2.6	37.20	-
Test Item	10	5.7	5	2.5	38.40	[1]
	100	5.0	4	2.5	37.50	2
	1000 R1	5.1	5	1.9	38.40	[1]
	1000 R2	5.6	6	1.8	38.00	0
	1000 R3	5.3	4	2.7	39.00	[2]
3,5dichlorophenol	3.2	7.4	10	3.7	22.20	42
	10	7.4	10	5.1	13.80	64
	32	8.1	10	7.5	3.60	91

R₁– R₄= Replicates 1 to 4

[Increase in respiration rate as compared to controls] 

 

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.

Executive summary:

A study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)".

Methods

Activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at measured temperatures of between 19 and 20 °C with the addition of a synthetic sewage as a respiratory substrate.

The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item, 3,5-dichlorophenol.

Results

The effect of the test item on the respiration of activated sewage sludge gave a 3-Hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.

It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

The reference item gave a 3-Hour EC50value of 4.8 mg/L, 95% confidence limits 3.5 to 6.6 mg/L.

Description of key information

A study was performed to assess the effect of the test item on the respiration of activated sewage sludge. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2010) No. 209 "Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)".

The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 1000 mg/L.  The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L