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Ecotoxicological information

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:
28 January to 03 April 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
This study was performed according to OECD Guideline 209 with GLP statement. All validity criteria were fulfilled.
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on 10 July 2012 / signed on 30 November 2012)
Analytical monitoring:
no
Details on sampling:
Not applicable
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
A mixed population of activated sludge micro-organisms was obtaines on 5 March 2014 from the aeration stage of the Severn trent Water Plc sewage plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.
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 sample was 8.0 measured using a Hach HQ40d Flexi handheld meter. Determination of the suspended solids level of teh activated sewage sludge was carried out by filtering a sample (100 mL) of the activated sewage sludge by suction through a pre-weihed 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 was equal to 3.0 g/L.
Test type:
static
Water media type:
other: deionized reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC)
Limit test:
no
Total exposure duration:
3 h
Hardness:
None
Test temperature:
The test was conducted under normal laboratory lighting in a temperature controlled room at approximately 20 °C.
pH:
The pH of the synthetic sewage stock was 7.1. The pH of the test item dispersions was meaured after stirring and adjusted between pH 7.0 and 8.0.
The pH of test preparations was measured at the test start and at the end of the 3-Hour incubation period using a Hach HQ40d Flexi handheld meter
Dissolved oxygen:
The oxygen concentrations in all vessels were measured after 30 minutes contact time.
Salinity:
None
Conductivity:
None
Nominal and measured concentrations:
As it was not a requirement for 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 GLP and has been reflected in the GLP compliance statement.
Details on test conditions:
AMount of test item (5,50, 500 mg (in triplicate)) were each separately dispersed in approximately 200 mL of deionized reverse osmosis water and subject to ultrasonisation for approximately 15 minutes followed by magnetic stirring for 24 hours in order to maximize the dissolved test item concentrtaions. 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 maintenained under identical conditions but not exposed to the test item.

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 (firts control). the mixture was aereted 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 was conducted under normal laboratory lighting in a temperature controlled room at approximately 20 °C.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
the dissolved oxygen concentrations after 30 minutes contact time in all vesseks were above 60 to 70% of the dissolved oxygen saturation level of 8.9 mgO2/L.
No statistically significant toxic effects were shown at the test concentration of 10 mg/L (p<0.05) however statistically significant toxic effects were shown at the test concentrations of 100 and 1000 mg/L.
The test item showed inhibition of 33% at 100 mg/l and greater than 91% at 1000 mg/L. Based on these results, the EC50 value for the test item is greater than 100 mg/L and after discussion with the Sponsor the EC50 value will be reported as such.
The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 100 mg/L.
Results with reference substance (positive control):
EC10(3hrs)=1.9 mg/L
EC20(3hrs)=2.7 mg/L
EC50(3hrs)=7.8 mg/L (CL: 6.1-9.9 mg/L)
EC80=23 mg/L
Nominal concentration (mg/L)  Initial Reading (mg O2/L)  Measurement period (min)  Final O2 redaing (mg O2/L)  O2 consumption rates (mg O2/L/Hour) % Inhibition 
 Control R1  4.1  3  1.8 46.0   -
 Control R2  4.3  3  2.0  46.0  -
 Control R3  4.5  3  2.3  44.0  -
 Control R4  5.1  4  2.2  43.5  -

Test item 10

 4.8

 4

 1.9

 43.50

 3

 Test item 100

 4.5

 4

 2.5

 30.00

33 

 Test item 1000 R1

 7.8

 5

 7.5

 3.60

 92

 Test item 1000 R2

 8.2

 10

 7.5

 4.20

 91

 Test item 1000 R3

 7.7

10

 7.0

 4.20

 91

 3,5-dichlorophenol 3.2  6.0  6  2.5  35.00  22
 3,5-dichlorophenol 10  7.1  10  4.3  16.80  63
 3,5-dichlorophenol 32  8.2  10 7.2   6.00  87

Table: pH values of the test preparation at the start and end of teh Exposure period in the Range-finding test

 Conditions  Nominal concentrations (mg/L) pH values 0 Hours   pH values 3 Hours
Control            R1  7.6 7.5 
 R2  7.6  7.5
 R3  7.7  7.5
 R4  7.7 7.5 
 Test item              10  7.7  7.5
 100  7.6  7.4
 1000 R1  7.6  7.8
 1000 R2  7.6  7.8
 1000 R3  7.6  7.8
 3,5 -dichlorophenol        3.2  7.6  7.8
 10  7.6 8.1 
 3.2  7.7  8.2
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 100 mg/L.
Validation criteria: the coefficient variation of oxygen uptake in the control vessels was 2.92% and the specific respiration rate of the controls was 29.92 mg oxygen per gram dry weight of sludge per hour. The validation criteria have there been satisfied. The validation criteria for the reference item EC50 value was also satisfied.
Executive summary:

One key study is available (Harlan, 2014) to assess the toxicity of the registered substance to microorganisms. In this study, the effect of the registered substance on the respiration rate of activated sludge was investigated after a contact time of 3 hours, according to OECD Guideline 209 with GLP statement.The test was carried out under static conditions with the test substance concentrations of 10, 100 and 1000 mg/L.

The inhibitory effects of the test and reference substances were determined in comparison to the control respiration rates.The mean inhibition of respiration for the test substance replicates were 3, 33 and 91.3 % at 10, 100 and 1000 mg/L, respectively.In order to check the activity of the test system and the test conditions a reference test was carried out with 3,5 -dichlorophenol as reference item and the reference toxicity was determined. The EC50-value for the reference item was 7.8 mg/L.This is in the recommended range of validity of 2 to 25 mg/L.

Under the test conditions, the effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 100 mg/L.

The reference item gave a 3 -Hour EC50 value of 7.8 mg/L (95% CL: 6.1 -9.9 mg/L)

This study is scored with a klimish value of 2 because two out of three concentrations tested (100 and 1000 mg/l) are higher than the water solubility (51.17 mg/L).

Description of key information

Based on OECD 209 GLP test, the effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 100 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
100 mg/L

Additional information

One key study is available (Harlan, 2014) to assess the toxicity of the registered substance to microorganisms. In this study, the effect of the registered substance on the respiration rate of activated sludge was investigated after a contact time of 3 hours, according to OECD Guideline 209 with GLP statement.The test was carried out under static conditions with the test substance concentrations 10, 100 and 1000 mg/L. The inhibitory effects of the test and reference substances were determined in comparison to the control respiration rates.The mean inhibition of respiration for the test substance replicates were 3, 33 and 91.3 % at 10, 100 and 1000 mg/L, respectively.In order to check the activity of the test system and the test conditions a reference test was carried out with 3,5 -dichlorophenol as reference item and the reference toxicity was determined. The EC50-value for the reference item was 7.8 mg/L.This is in the recommended range of validity of 2 to 25 mg/L.

Under the test conditions, the effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 value of greater than 100 mg/L.