N-(hydroxymethyl)acrylamide

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 06 August 2010 and 27 August 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.6800 (Modified Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N-(hydroxymethyl)acrylamide

EC Number:

213-103-2

EC Name:

N-(hydroxymethyl)acrylamide

Cas Number:

924-42-5

Molecular formula:

C4H7NO2

IUPAC Name:

N-(hydroxymethyl)acrylamide

Details on test material:

Sponsor's identification :Flocryl NMA 48
Description :clear colourless liquid
Batch number :LQ25A1796
Date received :28 July 2010
Expiry date :not supplied
Storage conditions :approximately 4ºC in the dark

A summary of the test item characterisation (performed at Harlan Laboratories Ltd.,Shardlow, UK) is given in Appendix 1 see in attached section.

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

- Concentrations:
concentrations of 3.2, 10, 32, 100, 320 and 1000 mg ai/l

- Sampling method:
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 and synthetic sewage. The pH of the control, reference item and test item preparations were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter at 0 hours and prior to measurement of the oxygen consumption rate after 3 hours contact time.

- Sample storage conditions before analysis:
not specified in report

Test solutions

Vehicle:

no

Details on test solutions:

The test item has a purity value of 42.8% (data supplied by the Sponsor). Therefore all test concentrations were corrected to account for this.
Based on the results obtained for a similar test on a similar test item activated sewage sludge micro-organisms in the definitive test were exposed to nominal test concentrations of 1.0, 3.2, 10, 32, 100, 320 and 1000 mg ai/l in order to ensure that a NOEC and possible an EC50 value for the test item were obtained.

For the purpose of the test, the test item was dispersed directly in water.
An amount of test item (4673 mg) was dissolved in water and the volume adjusted to 1 litre to give a 2000 mg ai/l stock solution from which dilutions were made to give 200, 20 and 2.0 mg ai/l stock solutions. An aliquot (250 ml) of the 2.0 mg ai/l stock solution was dispersed with synthetic sewage (16 ml), activated sewage sludge (200 ml) and water, to a final volume of 500 ml, to give the required concentration of 1.0 mg ai/l. Similarly, aliquots (80 and 250 ml) of the 20 and 200 mg ai/l stock solutions and aliquots (80 and 250 ml) of the 2000 mg ai/l stock solution were used to prepare the test concentrations of 3.2, 10, 32, 100, 320 and 1000 mg ai/l. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the stock solution.
The control group was maintained under identical conditions but not exposed to the test item.
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 has been reflected in the GLP compliance statement.

- Eluate:
At time "0" 16 ml of synthetic sewage was diluted to 300 ml with water and 200 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 litre per minute. Thereafter, at 15 minute intervals the procedure was repeated with appropriate amounts of the reference item being added. The test item vessels were prepared as described above. Finally a second control was 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 6.5 mg O2/l and 2.5 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.
The test was conducted under normal laboratory lighting in a temperature controlled room at 21±1ºC.


- Differential loading:
Not applicable.

- Controls:
The control group was maintained under identical conditions but not exposed to the test material.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
Not applicable.

- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)):
Not applicable.

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21ºC and was used on the day of collection. The pH of the sample was pH 7.3 and measured using a WTW pH/Oxi 340I pH. 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 deionised reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 4.0 g/l prior to use.

* rinsed three times with 20 ml deionised reverse osmosis water prior to drying in an oven.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

Not applicable.

Test conditions

Hardness:

The test water used for the test was laboratory tap water dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex water softener) giving water with a total hardness of approximately 140 mg/l as CaCO3. After dechlorination and softening the water was then passed through a series of computer controlled plate heat exchangers to achieve the required temperature. Typical water quality characteristics for the tap water as supplied, prior to dechlorination and softening, are given in Appendix 2 see in attached section.

Test temperature:

The test was conducted under normal laboratory lighting in a temperature controlled room at 21±1 Deg C.

pH:

The pH values of the test preparations at the start and end of the exposure period are given in Table 2 see in any other information on result section.

Dissolved oxygen:

The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between approximately 6.5 mg O2/l and 2.5 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

Salinity:

Not applicable.

Nominal and measured concentrations:

nominal test concentrations of 1.0, 3.2, 10, 32, 100, 320 and 1000 mg ai/l

Details on test conditions:

At time "0" 16 ml of synthetic sewage was diluted to 300 ml with water and 200 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 litre per minute. Thereafter, at 15 minute intervals the procedure was repeated with appropriate amounts of the reference item being added. The test item vessels were prepared as described in Section 3.4.1. Finally a second control was 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 6.5 mg O2/l and 2.5 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.
The test was conducted under normal laboratory lighting in a temperature controlled room at 21±1ºC.
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 and synthetic sewage. The pH of the control, reference item and test item preparations were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter at 0 hours and prior to measurement of the oxygen consumption rate after 3 hours contact time.


TEST MEDIUM / WATER PARAMETERS
Dechlorinated tap water

OTHER TEST CONDITIONS
- Adjustment of pH:
The pH values of the test preparations at the start and end of the exposure period are given in Table 2 see in any other information on result section.

- Photoperiod:
3 hours.

- Light intensity:
Normal laboratory lighting.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
In order to calculate the inhibitory effect of the test and reference materials the respiration rate was expressed as a percentage of the two control respiration rates.

% inhibition = [ 1 – 2 RS ] x 100
RC1 + RC2
where
RS = oxygen consumption rate for test or reference sample
RC1 + RC2 = oxygen consumption rates for controls 1 and 2
The percentage inhibition values were plotted against concentration for the reference item only, a line fitted using the Xlfit software package (IDBS) and the EC20, EC50 and EC80 values determined from the equation for the fitted line.
The EC20, EC50 and EC80 values for the test item were determined by inspection of the inhibition of respiration rate data.
95% confidence limits were calculated for the EC50 values for the reference item only using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon 1949).
The No Observed Effect Concentration (NOEC) was determined by inspection of the inhibition of respiration rate data.
The results of the study are considered valid if (i) the two control respiration rates are within 15% of each other and (ii) the EC50 (3-Hour contact time) for 3,5-dichlorophenol lies within the range 5 to 30 mg/l.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2


- Range finding study -NA for this study

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

2.29 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: not specified

Effect concentrations 2

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

71.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: not specified

Effect concentrations 3

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1.6 other: mg/l ai/l

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: not specified

Effect concentrations 4

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

50 other: mg/l ai/l

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: not specified

Details on results:

Oxygen consumption rates and percentage inhibition values for the control, test and reference items are given in Table 1 see in any other information. The pH values of the test preparations at the start and end of the exposure period are given in Table 2 see in any other information on result section, and observations made on the test preparations throughout the study are given in Table 3 see in any other information on result section.
Percentage inhibition is plotted against concentration for the test and reference items (Figures 1 to 2).
The following results were derived:
Flocryl NMA 48
3,5-dichlorophenol
ECx (3 Hours)
(mg ai/l) 95% Confidence Limits (mg ai/l) ECx (3 Hours)
(mg/l) 95% Confidence Limits (mg/l)
EC20 2.1 - 1.1 -
EC50 50 - 5.2 3.7 – 7.3
EC80 >1000 - 25 -
NOEC 1.6 - 0.8 -
It was not possible to determine an EC80 value for the test item as no concentration tested resulted in greater than 80% inhibition. It was also not possible to obtain 95% confidence limits for the test item EC50 value as the data generated did not fit the models available for calculation.
Variation in respiration rates of controls 1 and 2 after 3 hours contact time was ± 4%.
The validation criteria for the control respiration rates have been satisfied. The EC50 value for the reference item after 3 hours contact time 5.2 mg/l) was within the range of
5 –30 mg/l given in the validation criteria, although the 95% confidence limits for this value ranged from 3.7 – 7.3 mg/l. The lower end of this range was slightly below the range given for the EC50 value for the reference item after 3 hours contact time in the validation criteria. This was considered to have had no effect on the validity of the study given that the EC50 value for the reference item after 3 hours contact time was within the range stated in the validation criteria.

Results with reference substance (positive control):

Reference item preparation
For the purpose of the test a reference item, 3,5-dichlorophenol (Sigma-Aldrich Batch No. 0591OHJ) was used. Two stock solutions of 50 and 160 mg/l were prepared by dissolving the reference item directly in water with the aid of ultrasonication for approximately 20 minutes. Aliquots (10 and 100 ml) of the 160 mg/l stock solution were removed and dispersed with activated sewage sludge, synthetic sewage and water to give the final concentrations of 3.2 and 32 mg/l. Similarly, a 100 ml aliquot of the 50 mg/l stock solution was used to prepare the 10 mg/l concentration. The volumetric flasks containing the reference item were inverted several times to ensure homogeneity of the solutions.

Reported statistics and error estimates:

None.

Any other information on results incl. tables

Table1              Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time

Nominal Concentration		Initial O2 Reading (mg O2/l)	Measurent Period (minutes)	Final O2Reading (mg O2/l)	O2Consumption Rates (mg O2/l/min)	% Inhibition
Control	R1	6.7	10	3.0	0.37	-
	R2	6.7	10	2.7	0.40	-
Test Item	1.0 mg ai/l	6.7	10	2.9	0.38	1
	3.2 mg ai/l	7.7	10	5.1	0.26	32
	10 mg ai/l	7.6	10	5.5	0.21	45
	32 mg ai/l	7.6	10	5.6	0.20	48
	100 mg ai/l	7.7	10	5.8	0.19	51
	320 mg ai/l	7.9	10	5.9	0.20	48
	1000 mg ai/l	7.9	10	6.2	0.17	56
3,5-dichlorophenol	3.2 mg/l	7.4	10	5.1	0.23	40
	10 mg/l	8.0	10	6.6	0.14	64
	32 mg/l	8.5	10	7.9	0.06	84

 

R₁– R₂= Replicates 1 to 2

Table2              pH Values of the Test Preparations at the Start and End of the Exposure Period

Nominal Concentration		pH
		0 Hours	3 Hours
Control	R1	7.4	7.9
	R2	7.6	8.0
Test Item	1.0 mg ai/l	7.5	7.9
	3.2 mg ai/l	7.5	8.1
	10 mg ai/l	7.5	8.1
	32 mg ai/l	7.5	8.1
	100 mg ai/l	7.5	8.1
	320 mg ai/l	7.5	8.1
	1000 mg ai/l	7.5	8.0
3,5-dichlorophenol	3.2 mg/l	7.4	8.1
	10 mg/l	7.4	8.2
	32 mg/l	7.5	8.2

R₁– R₂= Replicates 1 to 2

Table3              Observations on the Test Preparations Throughout the Test Period

Nominal Concentration		Observations on Test Preparations
		0 Hours	30 Minutes Contact Ti	3 Hours Contact Ti
Control	R1	Dark brown dispersion	Dark brown dispersion	Dark brown dispersion
	R2	Dark brown dispersion	Dark brown dispersion	Dark brown dispersion
Test Item	1.0 mg ai/l	Clear colourless solution with no test item visible[*]	Dark brown dispersion with no undissolved test item visible	Dark brown dispersion with no undissolved test item visible
	3.2 mg ai/l	Clear colourless solution with no test item visible*	Dark brown dispersion with no undissolved test item visible	Dark brown dispersion with no undissolved test item visible
	10 mg ai/l	Clear colourless solution with no test item visible*	Dark brown dispersion with no undissolved test item visible	Dark brown dispersion with no undissolved test item visible
	32 mg ai/l	Clear colourless solution with no test item visible*	Dark brown dispersion with no undissolved test item visible	Dark brown dispersion with no undissolved test item visible
	100 mg ai/l	Clear colourless solution with no test item visible*	Dark brown dispersion with no undissolved test item visible	Dark brown dispersion with no undissolved test item visible
	320 mg ai/l	Clear colourless solution with no test item visible*	Dark brown dispersion with no undissolved test item visible	Dark brown dispersion with no undissolved test item visible
	1000 mg ai/l	Clear colourless solution with no test item visible*	Dark brown dispersion with no undissolved test item visible	Dark brown dispersion with no undissolved test item visible
3,5-dichlorophenol	3.2 mg/l	Dark brown dispersion with no undissolved reference item visible	Dark brown dispersion with no undissolved reference item visible	Dark brown dispersion with no undissolved reference item visible
	10 mg/l	Dark brown dispersion with no undissolved reference item visible	Dark brown dispersion with no undissolved reference item visible	Dark brown dispersion with no undissolved reference item visible
	32 mg/l	Dark brown dispersion with no undissolved reference item visible	Dark brown dispersion with no undissolved reference item visible	Dark brown dispersion with no undissolved reference item visible

R₁– R₂= Replicates 1 to 2

[*]Observations made prior to the addition of activated sewage sludge and synthetic sewage

Applicant's summary and conclusion

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 of 50 mg ai/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1.6 mg ai/l. Based on this, the EC50 at 3 hours for the test substance was71.4 mg/l and the NOEC was 2.29 mg/l.