C-M5

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 15 January 2008 and 22 January 2008.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 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 no effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994))

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

Analysis of the concentration, homogeneity and stability of the test material in the test preparations was not appropriate to the Test Guideline.

Test solutions

Vehicle:

no

Details on test solutions:

The test concentration to be used in the definitive test was determined by a preliminary range-finding test.
In the range-finding test activated sewage sludge micro-organisms were exposed to nominal test concentrations of 100 and 1000 mg/l. The test material was dissolved directly in water.

Based on the results of the range-finding test a "limit test" was conducted at a concentration of 1000 mg/l (three replicate vessels) to confirm that at this concentration no effect on respiration of the activated sewage sludge was observed.

Test material preparation:
For the purpose of the definitive test, the test material was dissolved directly in water.
An amount of test material (2000 mg) was dissolved in water with the aid of shaking by hand for approximately 1 minute. As the test material had not completely dissolved the stock was further subjected to ultrasonication for approximately 5 minutes and the volume adjusted to 1 litre to give a 2000 mg/l stock solution. An aliquot (250 ml) of this 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 1000 mg/l. The volumetric flask containing the stock solution was inverted several times to ensure homogeneity of the stock solution.
This method of preparation was performed for the three separate replicate test vessels.
The control group was maintained under identical conditions but not exposed to the test material.
Analysis of the concentration, homogeneity and stability of the test material in the test preparations was not appropriate to the Test Guideline.

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

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.

Preparation of 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 7.6 measured using a WTW pH/Oxi 340I 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 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.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

3 hours.

Test conditions

Hardness:

The test water used for both the range-finding and definitive tests 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.

Test temperature:

The test was conducted in a temperature controlled room at 21±1 deg C.

pH:

pH measured at 0 and 3 hours.
Control: 7.8 - 8.0
Test substance: 7.8 - 8.0
Reference substance: 7.7 - 8.3

Dissolved oxygen:

In some instances, the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines (6.5 mg O2/l and 2.5 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.

Salinity:

No data.

Nominal and measured concentrations:

Nominal test concentrations of 100 and 1000 mg/l in range finding test and 1000 mg/l in definitive test.

Analysis of the concentration, homogeneity and stability of the test material in the test preparations was not appropriate to the Test Guideline.

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flask.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 500 ml
- Aeration: 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
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable.
- Renewal rate of test solution (frequency/flow rate): Not applicable.
- No. of organisms per vessel: Not applicable.
- No. of vessels per concentration (replicates): 3 replicate vessels.
- No. of vessels per control (replicates): 2
- No. of vessels per reference control (replicates): 3 concentrations, 3.2, 10, 32 mg/l



OTHER TEST CONDITIONS
- Adjustment of pH: Not adjusted.
- Photoperiod: No data
- Light intensity: No data


EFFECT PARAMETERS MEASURED : dissolved oxygen (rate of respiration), pH


TEST CONCENTRATIONS

- Range finding study
- Test concentrations: 100 and 1000 mg/l
- Results used to determine the conditions for the definitive study: No significant effect on respiration was observed at either test concentration employed in the range finding study. Based on this information, a single test concentration, in triplicate, of 1000 mg/l was selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

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

Remarks:

respiration rate

Remarks on result:

other: 95% Cl limits not applicable

Effect concentrations 2

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

Remarks:

respiration rate

Remarks on result:

other: 95% CL limits not applicable.

Details on results:

Range-finding Test:
No significant effect on respiration was observed at either test concentration employed. Based on this information, a single test concentration, in triplicate, of 1000 mg/l was selected for the definitive test.

Definitive Test
Oxygen consumption rates and percentage inhibition values for the control, test and reference materials in the definitive test are given in Tables 2 and 3. The pH values of the test preparations at the start and end of the exposure period are given in Table 4, and observations made on the test preparations throughout the study are given in Table 5 (see remarks section for tables).

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

Results with reference substance (positive control):

EC20 (30 minutes) - 3.7 mg/l
EC20 (3 hours) - 3.7 mg/l

EC50 (30 minutes) - 12 mg/l
EC50 (3 hours) - 10 mg/l

EC80 (30 minutes) - >32 mg/l
EC80 (3 hours) - 29 mg/l

It was not possible to obtain an EC80 value after 30 minutes contact time for the reference material as no concentration tested resulted in greater than 80% inhibition. It was also not possible to obtain 95% confidence limits for the reference material EC50 values after 30 minutes or 3 hours contact time as the data generated did not fit the models available for calculation.
Variation in respiration rates of controls 1 and 2 after 30 minutes was ± 4%, and ± 3% after 3 hours contact time.
The validation criteria for the control respiration rates and reference material EC50 values were therefore satisfied.

Reported statistics and error estimates:

The percentage inhibition values were plotted against concentration for the reference material only, a line fitted using the Xlfit software package (IDBS) and the EC20, EC50 and EC80 (after 3 hours) values determined from the equation for the fitted line.
The EC20, EC50 and EC80 values for the test material and the EC80 value for the reference material after 30 minutes contact time were determined by inspection of the inhibition of respiration rate data.
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.

Any other information on results incl. tables

Table2               Oxygen Consumption Rates and Percentage Inhibition Values in the
Definitive Test after 30 Minutes Contact Time

Nominal Concentration (mg/l)		Initial O2 Reading (mg O2/l)	Measurement Period (minutes)	Final O2Reading (mg O2/l)	O2Consumption Rates (mg O2/l/min)	% Inhibition
Control	R1	5.8	6	2.5	0.55	-
	R2	5.5	3	3.7	0.60	-
Test Material	1000 R1	4.8	4	2.4	0.60	[4]
	1000 R2	5.3	3	3.5	0.60	[4]
	1000 R3	5.5	4	3.2	0.58	[1]
3,5-dichlorophenol	3.2	6.3	8	2.5	0.48	17
	10	7.2	10	4.0	0.32	44
	32	8.2	10	6.9	0.13	77

[Increase in respiration rate as compared to controls]

R₁– R₃= Replicates 1 to 3

Table3               Oxygen Consumption Rates and Percentage Inhibition Values in the
Definitive Test after 3 Hours Contact Time

Nominal Concentration (mg/l)		Initial O2 Reading (mg O2/l)	Measurement Period (minutes)	Final O2Reading (mg O2/l)	O2Consumption Rates (mg O2/l/min)	% Inhibition
Control	R1	6.7	8	2.3	0.55	-
	R2	5.9	6	2.8	0.52	-
Test Material	1000 R1	5.9	4	3.6	0.58	[8]
	1000 R2	5.5	5	2.8	0.54	[1]
	1000 R3	6.0	6	2.7	0.55	[3]
3,5-dichlorophenol	3.2	6.7	9	2.6	0.46	14
	10	7.8	10	5.3	0.25	53
	32	8.6	10	7.6	0.10	81

[Increase in respiration rate as compared to controls]

R₁– R₃= Replicates 1 to 3

Table4               pH Values of the Test Preparations at the Start and End of the Exposure Period

Nominal Concentration (mg/l)		pH
		0 Hours	3 Hours
Control	R1	7.8	8.0
	R2	7.8	8.0
Test Material	1000 R1	7.8	8.0
	1000 R2	7.8	8.0
	1000 R3	7.8	8.0
3,5-dichlorophenol	3.2	7.7	8.1
	10	7.7	8.2
	32	7.7	8.3

R₁– R₃= Replicates 1 to 3

Table5               Observations on the Test Preparations Throughout the Test Period

Nominal Concentration (mg/l)		Observations on Test Preparations
		0 Hours	30 Minutes Contact Time	3 Hours Contact Time
Control	R1	Dark brown dispersion	Dark brown dispersion	Dark brown dispersion
	R2	Dark brown dispersion	Dark brown dispersion	Dark brown dispersion
Test Material	1000 R1	Very dark red dispersion, no undissolved test material visible	Very dark red dispersion, no undissolved test material visible	Very dark red dispersion, no undissolved test material visible
	1000 R2	Very dark red dispersion, no undissolved test material visible	Very dark red dispersion, no undissolved test material visible	Very dark red dispersion, no undissolved test material visible
	1000 R3	Very dark red dispersion, no undissolved test material visible	Very dark red dispersion, no undissolved test material visible	Very dark red dispersion, no undissolved test material visible
3,5-dichlorophenol	3.2	Dark brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible
	10	Dark brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible
	32	Dark brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible	Dark brown dispersion, no undissolved reference material visible

 

R₁– R₃= Replicates 1 to 3

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

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

Executive summary:

Introduction.

A study was performed to assess the effect of the test material on the respiration of activated sewage sludge. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 209 "Activated Sludge, Respiration Inhibition Test", Method C11 of EEC Commission Directive 88/302/EEC and US EPA Draft Ecological Effects Test Guidelines OPPTS 850.6800.

Methods.

Following a preliminary range-finding test, activated sewage sludge was exposed to an aqueous solution of the test material at a concentration of 1000 mg/l (three replicate flasks) for a period of 3 hours at a temperature of approximately 21°C with the addition of a synthetic sewage as a respiratory substrate.

The rate of respiration was determined after 30 minutes and 3 hours contact time and compared to data for the control and a reference material, 3,5-dichlorophenol.

Results.

The effect of the test material on the respiration of activated sewage sludge gave a 3‑Hour EC₅₀of greater than 1000 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was1000mg/l.

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

The reference material gave a 3-Hour EC₅₀value of 10 mg/l.