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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Phase: 31 January 2018. Report issued: 16 July 2018.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Based on the results of a range-finding test the following test concentrations were assigned to the definitive test: 10, 100 and 1000 mg/L.

A nominal amount of test item (2500 mg) was dissolved in water with the aid of ultrasonication for approximately 30 minutes, followed by vigorous shaking by hand and the volume adjusted to 1 litre to give a 2500 mg/L stock solution from which dilutions were made to give the required test substance concentrations.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: A mixed population of activated sewage sludge micro-organisms was obtained on 30 January 2018 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 exposure: 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 (9.5 litres) was fed synthetic sewage (475 mL). The pH of the sample on the day of the test was 7.6 measured using a pH meter
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Post exposure observation period:
None
Test temperature:
20 - 21 °C
pH:
7.3 - 8.0 (definitive test)
Dissolved oxygen:
6.34 - 8.0 mg O2/L (definitive test)
Nominal and measured concentrations:
Test concentrations were 10, 100 and 100 mg/L of test susbtance based on nominal concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 ml conical flasks
- Aeration: continuous aeration
- No. of vessels per concentration (replicates): Triplicate for 1000 mg/L concentration, one each for 100 and 10 mg/L concentrations.
- No. of vessels per control (replicates): four
- Sludge concentration (weight of dry solids per volume): 3.0 g/L prior to use

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test water used for the test was deionised reverse osmosis water containing less than 1 mg/L Total Organic Carbon (TOC).

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the test item stock solutions were measured using a handheld meter and if necessary, adjusted to between pH 7.0 to pH 8.0.
- Photoperiod: The test was conducted under normal laboratory lighting
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
< 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other:
Remarks:
Experimental work indicated that the test item was volatile in solution as when a steady stream of compressed air (0.5 litres per minute) was bubbled through a solution of the test item over three hours, a 88% loss of test item occurred. This was determined by Dissolved Organic Carbon analysis. Therefore the NOEC value for the test item is considerd to be an underestimate and not relevant for use in assessment of actual toxicity to sludge microorganisms in the field.
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Basis for effect:
inhibition of total respiration
Remarks on result:
other:
Remarks:
Experimental work indicated that the test item was volatile in solution as when a steady stream of compressed air (0.5 litres per minute) was bubbled through a solution of the test item over three hours, a 88% loss of test item occurred. This was determined by Dissolved Organic Carbon analysis. Therefore the NOEC value for the test item is considerd to be an underestimate and not relevant for use in assessment of actual toxicity to sludge microorganisms in the field.
Results with reference substance (positive control):
The EC50 value (3-Hour contact time) for the reference item, 3,5-Dichlorophenol, was 8.2mg/L.
Reported statistics and error estimates:
95% confidence limits were calculated for the reference item EC50 value using the method of Litchfield and Wilcoxon (Litchfield and Wilcoxon, 1949).

One way analysis of variance incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison procedure for comparing several treatments with a control was carried out on the oxygen consumption data after 3 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

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.

Statistically significant toxic effects were shown at all the test concentrations employed.

Based on these results it was considered unnecessary to perform a definitive test.

The results for inhibition of respiration are presented in the following table:

      Bromform (EC200 -854 -6)     3,5 -Dichlorophenol (postive control)   
 ECx (3 hours) (mg/L)*  95% Confidence Limits (mg/L)   ECx (3 hours) (mg/L)   95% Confidence Limits (mg/L)
 EC10  <10*  -  1.8  -
 EC20  <10*  -  2.7  -
 EC50  >1000*  -  8.2  6.4 - 11
 EC80  >1000*  -  25  -
 NOEC  <10  -  -  -

* Experimental work indicated that the test item was volatile in solution as when a steady stream of compressed air (0.5 litres per minute) was passed with a solution of the test item over three hours, a 88% loss of test item occurred. This was determined by Total Organic Carbon analysis. Therefore the

3 -hour ECx values and NOEC for the test item may be underestimates.

Validity criteria fulfilled:
yes
Conclusions:
The test substance gave a No Observed Effect Concentration (NOEC) after 3 hours exposure was less than 10 mg/L. However, analysis during the test indicated that the test item was volatile in solution as when a steady stream of compressed air (0.5 litres per minute) was bubbled through a solution of the test item over three hours, a 88% loss of test item occurred. Therefore the 3-Hour EC50 value for the test item is considered to be underestimate and not relevant for use in assessment of actual toxicity to sludge microorganisms in the field.
Executive summary:

Introduction

The study performed to assess the effect of the test substance 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/L (three replicates of the 1000 mg/L test concentration) for a period of 3 hours at a measured temperature of between 20 °C and 21 °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 and Conclusion

The test substance gave a No Observed Effect Concentration (NOEC) after 3 hours exposure was less than 10 mg/L. However, analysis during the test indicated that the test item was volatile in solution as when a steady stream of compressed air (0.5 litres per minute) was passed with a solution of the test item over three hours, a 88% loss of test item occurred. Therefore the 3-Hour EC50 value for the test item is considered to be underestimate and not relevant for use in assessment of actual toxicity to sludge microorganisms in the field.


The reference item gave a 3-Hour EC50 value of 8.2 mg/L, 95% confidence limits 6.4 to 11 mg/L.



Description of key information

Toxicity of Bromoform (EC 200 -854 -6) to sewage sludge microorganisms.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
10 mg/L

Additional information

The test substance gave a 3-hour EC50 > 1000 mg/L and a 3 -hour No Observed Effect Concentration (NOEC) of < 10 10 mg/L.

Analysis during the test indicated that the test item was volatile in solution as when a steady stream of compressed air (0.5 litres per minute) was bubbled through a solution of the test item over three hours, a 88% loss of test item occurred. Therefore the 3-Hour EC50 and NOEC value for the test substance are considered to be underestimated as a significant proportion of the test substance vapourised from the test medium. As rapid volatisation and loss of the test substance can be expected to also occur in a waste water treatment plant, the 3-hour EC50 and NOEC values are not considered relevant for use in assessment of actual toxicity to sludge microorganisms, as little or no test substance is expected to be present in the waters within the treatment plant.