N-isopropylmethacrylamide

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:

Experimental Starting Date: 29 May 2012, Experimental Completion Date: 11 June 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

- Concentrations:
10, 100 and 1000 mg active ingredient (ai)/L

- Sampling method:
As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 ml darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe.

Vehicle:

no

Details on test solutions:

PREPARATION OF TEST SOLUTION

RANGE-FINDING TEST TO MEASURE TOTAL RESPIRATION:
For this range-finding test activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg active ingredient (ai)/L. The test item was dissolved directly in water. The test item contains 8.7% water therefore all test concentrations were corrected for this.

An amount of test item (2738 mg) was dissolved in water with the aid of high shear mixing at approximately 7500 rpm for approximately 30 minutes and the volume adjusted to 1 liter to give a 2500 mg ai/L stock solution from which dilutions were made to give 250 and 25 mg ai/L stock solutions. An aliquot (200 mL) of the 25 mg ai/L stock solution was dispersed with synthetic sewage (16 mL), activated sewage sludge (250 mL) and water, to a final volume of 500 mL, to give the required concentration of 10 mg ai/L. Similarly, aliquots (200 mL) of the 250 mg ai/L nd 2500 mg ai/L stock solutions were used to prepare the test concentrations of 100 and 000 mg ai/L. The 1000 mg ai/L test concentration was prepared in triplicate. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the stock solutions.

The pH of the test item stock solutions were measured using a WTW pH/Oxi 3401 pH and dissolved oxygen meter (see Table 1 - attached background material) and adjusted to between pH 7.0 to pH 8.0 using 1.0 M Sodium Hydroxide solution or 0.5 M Sulphuric acid solution.

The control group was maintained under identical conditions but not exposed to the test item.

RANGE-FINDING TEST TO MEASURE HETEROTROPHIC RESPIRATION:
For this range-finding test activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg active ingredient (ai)/L. The test item was dissolved directly in water. The test item contains 8.7% water therefore all test concentrations were corrected for this.

An amount of test item (2738 mg) was dissolved in water with the aid of high shear mixing at approximately 7500 rpm for approximately 30 minutes and the volume adjusted to 1 liter to give a 2500 mg ai/L stock solution from which dilutions were made to give 250 and 25 mg ai/L stock solutions.
An aliquot (200 mL) of the 25 mg ai/L stock solution was dispersed with synthetic sewage (16 mL), allythiourea (ATU) (5 mL of a 1.16 g/L stock solution), activated sewage sludge (250 mL) and water, to a final volume of 500 mL, to give the required concentration of 10 mg ai/L. Similarly, aliquots (200 mL) of the 250 mg ai/L and 2500 mg ai/L stock solutions were used to prepare the test concentrations of 100 and 1000 mg ai/L. The 1000 mg ai/L test concentration was prepared in triplicate. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the stock solutions.

The pH of the test item stock solutions were measured using a WTW pH/Oxi 3401 pH and dissolved oxygen meter (see Table 6 - see attached background material) and adjusted to between pH 7.0 to pH 8.0 using 1.0 M Sodium Hydroxide solution or 0.5 M Sulphuric acid solution.

The control group was maintained under identical conditions but not exposed to the test item.

PREPARATION OF REFERENCE ITEM
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 20 minutes. The pH of this stock solution was measured to be pH 5.1 and was adjusted to pH 7.1 using 1.0 M NaOH. The pH values were measured using a WTW pH/Oxi 3401 pH and dissolved oxygen 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 give the final 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:

TEST SPECIES:
A mixed population of activated sewage sludge micro-organisms was obtained on 29 May 2012 for the range-finding tests and 11 June 2012 for the definitive tests 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:
For both range-finding tests the activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21°C and was used on the day of collection. The pH of the sample used to measure total respiration was 7.4 and the pH of the sample used to measure heterotrophic respiration was 7.5. The pH values were measured using a WTW pH/Oxi 3401 pH and dissolved oxygen 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°C 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 for both range-finding tests was equal to 3.0 g/L prior to use.

SYNTHETIC SEWAGE:
A synthetic sewage of the following composition, was added to each test vessel to act as a respiratory substrate:
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 1 litre of water with the aid of ultrasonication.
In the range-finding test for total respiration, the pH of the synthetic sewage stock was pH 7.1 and in the range-finding test for heterotropic respiration the pH of the synthetic sewage stock was 6.9 and adjusted to pH 7.1 using 1.0 M NaOH. The pH values were measured using a WTW pH/Oxi 3401 pH and dissolved oxygen meter.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

Not applicable.

Hardness:

Not stated.

Test temperature:

The test was conducted under normal laboratory lighting in a temperature controlled room at 20±2°C.

pH:

The pH of the control, reference item and test item preparations was measured at 0 hours and prior to measurement of the oxygen consumption rate after 3 hours contact time.

Dissolved oxygen:

The oxygen concentrations in all vessels were measured after 30 minutes contact time.

Nominal and measured concentrations:

Nominal test concentrations in range-finding test to measure total respiration and range-finding test to measure heterotrophic respiration were:
10, 100 and 1000 mg (ai)/L.

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 approximately 0.5 - 1 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 in 'details of test solutions' section. Finally two further control vessels were prepared.

As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 ml darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between approximately 7.0 mg O2/l and 2.0 mg O2/l). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period.

Observations were made on the test preparations throughout the test period. Observations of the test item vessels at 0 hours were made prior to addition of activated sewage sludge.


TEST MEDIUM / WATER PARAMETERS
The test water used for the range-finding tests was deionized reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The rates of respiration (total respiration, heterotrophic respiration and nitrification respiration) were determined after 3 hours contact time and compared to data for the control and a reference item,3,5-dichlorophenol.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 other: mg (ai)/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: total respiration rate

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1 000 other: mg (ai)/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: heterotropic respiration and nitrification respiration

Details on results:

RANGE-FINDING TESTS

TOTAL RESPIRATION:
Oxygen consumption rates and percentage inhibition values for the control, test and reference items are given in Table 3. The pH values of the test preparations at the start and end of the exposure period are given in Table 4. Observations made on the test preparations throughout the study are given in Table 5. The dissolved oxygen concentrations in all vessels after 30 minutes contact time are given in Table 2.
See attached background material for all tables.

The coefficient of variation of oxygen uptake in the control vessels was 2.7% and the specific respiration rate of the controls was 24.33 mg oxygen per gram dry weight of sludge per hour. The validation criteria have therefore been satisfied.

In some instances, the initial and final dissolved oxygen concentrations were outside those recommended in the test guidelines (7.0 mg O2/L and 2.0 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.

It was not possible to determine an EC50 value for the test item as no concentration tested resulted in greater than 50% inhibition.

The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60 to 70% of the dissolved oxygen saturation level of 8.9 mg O2/L with the exception of control replicate R1 showing 59% saturation. This deviation was considered to have had no adverse effect on the study given that the oxygen consumption value was measured/calculated over the linear portion of the trace.

No statistically significant toxic effects were shown at the test concentrations of 10 and 100 mg ai/L (P ≥ 0.05), however statistically significant toxic effects were shown at the test concentration of 1000 mg ai/L (P< 0.05).

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

HETEROTROPIC RESPIRATION:
Oxygen consumption rates and percentage inhibition values for the control, test and reference items are given in Table 8. The pH values of the test preparations at the start and end of the exposure period are given in Table 9. Observations made on the test preparations throughout the study are given in Table 10. The dissolved oxygen concentrations in all vessels after 30 minutes contact time are given in Table 7.
See attached background material for all tables.

The coefficient of variation of oxygen uptake in the control vessels was 6.7%. The validation criteria have therefore been satisfied.

The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60 to 70% of the dissolved oxygen saturation level of 8.9 mg 02/L.

No statistically significant toxic effects were shown at all of the test concentrations employed. The NOEC can therefore be reported as being 1000 mg ai/L for heterotropic respiration.

NITRIFICATION RESPIRATION:
Oxygen consumption rates and percentage inhibition values for the control, test and reference items are given in Table 11 (see attached background material).

The coefficient of variation of oxygen uptake in the control vessels was 7.0%. The validation criteria have therefore been satisfied.

No statistically significant toxic effects were shown at all ofthe test concentrations employed. The NOEC can therefore be reported as being 1000 mg ai/L for nitrification respiration.

Results with reference substance (positive control):

TOTAL RESPIRATION:
Percentage inhibition is plotted against concentration for the reference item, 3,5-dichlorophenol (Figure 1 - see attached background material).

The following results were derived:
3 hour EC20: 2.9 mg/L
3 hour EC50: 8.1 mg/L (95% Confidence Limits: 6.4 - 10 mg/L)
3 hour EC80: 23 mg/L

The validation criterion for the reference item EC50 value was also satisfied.

HETEROTROPIC RESPIRATION:
Percentage inhibition is plotted against concentration for the reference item, 3,5-dichlorophenol (Figure 2 - see attached background material).

The following results were derived:
3 hour EC20: 7.2 mg/L
3 hour EC50: 20 mg/L (95% Confidence Limits: 16 - 25mg/L)
3 hour EC80: 56 mg/L

The validation criterion for the reference item EC50 value was also satisfied.

NITRIFICATION RESPIRATION:
Percentage inhibition is plotted against concentration for the reference item, 3,5-dichlorophenol (Figure 3 - see attached background material).

3 hour EC20: 1.5 mg/L
3 hour EC50: 4.3 mg/L (95% Confidence Limits: 3.4 - 5.4 mg/L)
3 hour EC80: 12 mg/L

The validation criterion for the reference item EC50 value was also satisfied.

Discussion:

Based on the results of the two range-finding tests, it was considered not necessary to perform a definitive test in order to obtain a total respiration NOEC value for the test item.

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the total respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 1000 mg ai/L.

Executive summary:

Introduction

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 solution of the test item at concentrations of 10, 100 and 1000 mg active ingredient (ai)/L (3 replicates of the 1000 mg ai/L test concentration) for a period of 3 hours at a temperature of 20 ± 2 °C with the addition of a synthetic sewage as a respiratory substrate and with and without the presence of specific nitrification inhibitor ATU.

The rates of respiration (total respiration, heterotrophic respiration and nitrification respiration) were 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 total respiration of activated sewage sludge gave a 3-Hour EC₅₀ value of greater than 1000 mg (ai)/L.

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

The reference item gave the following 3-Hour EC₅₀ values and 95% confidence limits.

	Total Respiration		Heterotropic Respiration		Nitrification Respiration
	ECx (3 Hours) (mg/L)	95% Confidence Limits (mg/L)	ECx (3 Hours) (mg/L)	95% Confidence Limits (mg/L)	ECx (3 Hours) (mg/L)	95% Confidence Limits (mg/L)
EC20	2.9	-	7.2	-	1.5
EC50	8.1	6.4 – 10	20	16 – 25	4.3	3.4 – 5.4
EC80	23	-	56	-	12

Description of key information

The effect of the test item on the total respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 1000 mg ai/L.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

Additional information

Introduction

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 solution of the test item at concentrations of 10, 100 and 1000 mg active ingredient (ai)/L (3 replicates of the 1000 mg ai/L test concentration) for a period of 3 hours at a temperature of 20 ± 2 °C with the addition of a synthetic sewage as a respiratory substrate and with and without the presence of specific nitrification inhibitor ATU.

The rates of respiration (total respiration, heterotrophic respiration and nitrification respiration) were 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 total respiration of activated sewage sludge gave a 3-Hour EC₅₀value of greater than 1000 mg (ai)/L.

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

It was considered not necessary to perform a definitive test in order to obtain a total respiration NOEC value for the test item.

The reference item gave the following 3-Hour EC₅₀values and 95% confidence limits.

	Total Respiration		Heterotropic Respiration		Nitrification Respiration
	ECx (3 Hours) (mg/L)	95% Confidence Limits (mg/L)	ECx (3 Hours) (mg/L)	95% Confidence Limits (mg/L)	ECx (3 Hours) (mg/L)	95% Confidence Limits (mg/L)
EC20	2.9	-	7.2	-	1.5
EC50	8.1	6.4 – 10	20	16 – 25	4.3	3.4 – 5.4
EC80	23	-	56	-	12