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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:
November 2016 - July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Nominal amounts of test item (5, 50 and 500 mg (in triplicate for 500 mg)) were each separately weighed onto a filter paper* prior to being dispersed in approximately 200 mL of deionized reverse osmosis water and subjected to ultrasonication for approximately 15 minutes followed by magnetic stirring for 24 hours, at temperatures of between 20°C and 21°C, in order to maximize the dissolved test item concentration.

Single test vessel replicates were prepared for the two lower concentrations and three replicates were prepared for the highest test item concentration. 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 (three replicates of the 1000 mg/L test concentration).

The pH of the test item dispersions was measured after stirring using a Hach HQ40d Flexi handheld meter.

A filter paper* was added to each control vessel in order to maintain consistency between the test and procedure control vessels.
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 overnight prior to use in the test. On the day of collection the activated sewage sludge (4 liters) was fed synthetic sewage (200 mL).
The pH of the sample on the day of the test was 7.7, measured using a Hach HQ40d Flexi handheld meter. Determination of the mixed liquor suspended solids level (MLSS or SS) of the activated sewage sludge was carried out on the day of collection prior to the addition of the synthetic sewage sludge and after being aerated overnight 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 three 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 ºC for at least one hour and allowed to cool before weighing. This process was repeated until two consecutive dry weights within 4% were attained. The SS concentration was equal to 3.0 g dry weight/L prior to use. The level of the SS in the final test medium after dilution was equivalent 1.5 g (dry weight)/L.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
The temperatures recorded in the first and last control vessels were measured to be approximately 21°C.
pH:
between pH 7.0 and 8.0.
Dissolved oxygen:
Ranged from 5.5 to 8.0 mg O2/L.
Nominal and measured concentrations:
10, 100 and 1000 mg/L.
Details on test conditions:
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 continuously with clean, oil-free compressed air via narrow bore glass tubes at a rate of 0.5 to 1.0 liter per minute. Thereafter, at 15 minute intervals the procedure was repeated for the second control followed by the reference item vessels (in ascending concentration order) with appropriate amounts of the reference item being added.

Finally two further control vessels were prepared. The addition of the inoculum to the reaction mixture was considered the start of the 3-hour exposure period for each vessel.

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 a final volume of 1 liter of water with the aid of ultrasonication.

Observations
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.The temperature was recorded in one of the control vessels at the start and end of the incubation period.

pH Measurements
The pH of test preparations was measured at the test start (i.e. after the addition of activated sludge) and at the end of the 3-hour incubation period.

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

Measurement of the Respiration Rates
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 7 mg O2/L and 2 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.
Reference substance (positive control):
yes
Key result
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
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
10 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
No statistically significant toxic effects were shown at all of the test concentrations employed (P > 0.05). It was therefore considered justifiable not to perform a definitive test. It was considered unnecessary to test at concentrations in excess of 1000 mg/L.
Results with reference substance (positive control):
The EC50 value for the reference item (8.3 mg/L) also satisfied the validation criterion.

Validation Criteria


The coefficient of variation of oxygen uptake in the control vessels was 3.2% and the specific respiration rate of the controls was 28.80 mg oxygen per gram dry weight of sludge per hour. The validation criteria have therefore been satisfied.


 


Results


 


Inhibition of Respiration Rate


 













































 



 


Test Item



 


3,5-dichlorophenol



3-hour EffectConcentrations(mg/L)



 


95% Confidence Limits (mg/L)



3-hour EffectConcentrations(mg/L)



 


95% Confidence Limits (mg/L)



EC10



>1000



-



1.7



 



EC20



>1000



-



2.5



 



EC50



>1000



-



8.3



6.3 - 11



NOEC



1000



-



-



-


Validity criteria fulfilled:
yes
Conclusions:
No effects of the test item were observed on the respiration of activated sewage sludge following a 3-hour exposure, resulting in an EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) following the 3-hour exposure was 1000 mg/L.
Executive summary:

A study following GLP was performed to assess the effect of the test item (EC =953-378-2) 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)".


 


Activated sewage sludge was exposed to an aqueous dispersion of the test item at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L test concentration) for a period of 3 hours at measured temperatures of approximately 21 °C with the addition of a synthetic sewage as a respiratory substrate. A reference item, 3,5-dichlorophenol, and control group were incubated concurrently and the rates of respiration were determined following the exposure period of 3 hours.


No effects of the test item were observed on the respiration of activated sewage sludge following a 3-hour exposure, resulting in an EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) following the 3-hour exposure was 1000 mg/L.


 


The 3-hour EC50 value for the reference item, 3,5-dichlorophenol, was determined to be 8.3 mg/L, with 95% confidence limits 6.3 to 11 mg/L.

Description of key information

An experimental study was carried out to determine the aquatic toxicity of the registered substance on activated sluge microorganisms according to the TG OECD 209 and GLP.


No effects of the test item were observed on the respiration of activated sewage sludge following a 3-hour exposure, resulting in an EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) following the 3-hour exposure was 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

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