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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:
05 July 2017 - 07 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:
22 July 2010
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
The test item is irritant or corrosive.
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L. Therefore, weighed amounts were added to testbottles containing 200 mL Milli-RO water. These mixtures were magnetically stirred for a short period of 5 minutes and subsequently, 16 mL synthetic medium made up to 50 mL with Milli-RO water and 250 mL sludge were added to the mixtures resulting in the required loading rates. Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring.

- Controls:
Blank control: medium with sludge and without test item
Abiotic control: medium without sludge and with the test item
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's- Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Preparation of inoculum for exposure: The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3.0 g/L of sludge, as used for the test).
- Pretreatment: The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium (=sewage feed) was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use. The initial pH of the sludge was 5.8 on the day of testing. The sludge was buffered to a pH of 7.5 using a 70 g/L sodium bicarbonate solution. Before the sludge was used the respiration rate of the sludge was determined to verify the quality of the sludge. The mean respiration rate was 24 mg O2/g.h and therefore, within the validity criteria.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
19.7 - 20.3°C
pH:
Start: 7.6-7.7
End: 7.3-8.1
Dissolved oxygen:
60-70% saturation at study initiation
Nominal and measured concentrations:
Nominal concentrations: 10, 100 and 1000 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: all glass open bottles/vessels, fill volume: 500 mL
- Aeration: yes, the aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the sludge flocs in suspension.
- No. of vessels per concentration (replicates): 3 for the highest loading rate (1000 mg/L) and 1 for the lower loading rates (10 and 100 mg/L)
- No. of vessels per control (replicates): 6
- No. of vessels per abiotic control (replicates): 1
- Sludge concentration (weight of dry solids per volume): 3.0 g/L after collection; 1.5 g/L in the final test solutions
- Nutrients provided for bacteria: synthetic sewage feed

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water (tap water purified by reverse osmosis)

OTHER TEST CONDITIONS
- Adjustment of pH: the initial pH of the sludge was adjusted before the preparation of the test solutions, the pH was not adjusted in the final test solutions.

EFFECT PARAMETERS MEASURED: inhibition of respiration at t=3 h

TEST CONCENTRATIONS
- Range finding study: 10, 100 and 1000 mg/L
- Results used to determined concentratins for the definitive test: since no inhibition of respiration rate was observed, the results of the combined limit/range-finding study were used to determine the effect parameters.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
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
Key result
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
Details on results:
- The results of the abiotic control showed that there was no oxygen uptake from abiotic processes.
- The combined limit/range-finding test showed 6% and no inhibition of the respiration rate at a loading rate of 10 and 100 mg/L, respectively. An average of 16% inhibition of the respiration rate was observed at 1000 mg/L. Therefore, the EC50 was above the highest loading rate tested (1000 mg/L).
- No statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 1000 mg KY-AF per litre.
- The reference substance showed that the used batch of sludge had normal sensitivity.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Concentrations tested (nominal): 1.0, 3.2, 10 and 32 mg/L
- Relevant effect levels: EC50: 4.15 mg/L (95% CI: 0.85-7.41 mg/L)
- Other: result was within the accepted range of 2 to 25 mg/L for total respiration.
Reported statistics and error estimates:
EC50 determination:
For the reference item calculation of the EC50 value was based on a 3-parameter logistic cumulative distribution function (CDF) using a non-linear regression analysis with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the reference item.
For the test item no EC50 value could be calculated because the test item proved to be non toxic up to and including the highest test concentration.

NOEC determination:
An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the control revealed significant inhibition of the respiration rate (Two-sample t-test Procedure, α=0.05, one-sided, smaller).

All calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).
Validity criteria fulfilled:
yes
Remarks:
see 'overall remarks' section for details
Conclusions:
Based on the results of a respiration inhibition test, performed according to OECD 209 (2010) and GLP principles, the 3h-NOEC and the 3h-EC50 for KY-AF were 1000 mg/L and >1000 mg/L, respectively.
Executive summary:

A respiration inhibition test was performed, according to OECD 209 (2010) and GLP principles, to assess whether KY-AF was toxic to wastewater bacteria. The test item was tested in a limit/dose-range finding test at nominal concentrations of 10, 100 and 1000 mg/L for an exposure period of 3 hours. The highest concentration was tested in triplicate, the lower concentrations were tested in one replicate. Six blank controls and one abiotic control were included in the test.

At 10 mg/L, an inhibition of 6.25% was observed, while at 100 mg/L no inhibition was observed. An average of 16% inhibition of the respiration rate was observed at 1000 mg/L. Therefore, the 3h-EC50 was above the highest loading rate tested (1000 mg/L). As the inhibition of the respiration rate at 1000 mg/L was not statistically significant, the 3h-NOEC was determined to be 1000 mg/L.

There was no oxygen uptake from abiotic processes. Testing with 3,5-dichlorophenol as reference item showed the test system had normal sensitivity. All validity criteria were met and the study was considered to be valid.

Description of key information

A reliable study performed according to OECD guideline 209 (2010) and GLP principles resulted in an 3h-EC50 of >1000 mg/L and a 3h-NOEC of 1000 mg/L.

A previous study, performed according to OECD guideline 209 (1984) and GLP principles, resulted in an 30min-EC50 of >100 mg/L and a 30min-NOEC of 100 mg/L, these results are considered supportive to the key results.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

Key study:

A respiration inhibition test was performed, according to OECD 209 (2010) and GLP principles, to assess whether KY-AF was toxic to wastewater bacteria. The test item was tested in a limit/dose-range finding test at nominal concentrations of 10, 100 and 1000 mg/L for an exposure period of 3 hours. The highest concentration was tested in triplicate, the lower concentrations were tested in one replicate. Six blank controls and one abiotic control were included in the test.

At 10 mg/L, an inhibition of 6.25% was observed, while at 100 mg/L no inhibition was observed. An average of 16% inhibition of the respiration rate was observed at 1000 mg/L. Therefore, the 3h-EC50 was above the highest loading rate tested (1000 mg/L). As the inhibition of the respiration rate at 1000 mg/L was not statistically significant, the 3h-NOEC was determined to be 1000 mg/L.

There was no oxygen uptake from abiotic processes. Testing with 3,5-dichlorophenol as reference item showed the test system had normal sensitivity. All validity criteria were met and the study was considered to be valid.

Supportive study:

This study was performed to assess the influence of the test item on the respiration rate of activated sludge after a contact time of 30 minutes. The study procedures were based on OECD guideline No. 209 (1984) and performed under GLP conditions. The test substance was hardly soluble in water and was quantitatively added to the test vessels at a concentration of 100 mg/L. During the 30 minute exposure period the test solution was continuously aerated, using a pipette as an aeration device. Oxygen consumption was recorded after 30 minutes for a period of approximately 10 minutes. During this period, the test solution was not aerated but continuously stirred on a magnetic stirrer. Exposure of the activated sludge to the test concentration of 100 mg per litre resulted in 12% inhibition of respiration in the first replicate and 6% inhibition in the second replicate. Average inhibition of both replicates was calculated as 9% (< 10% and therefore determined to be not significant). Since no significant effect was recorded at this concentration, no other concentrations were tested.

The batch of activated sludge was tested for sensitivity with the reference item 3,5-dichlorophenol. The EC50 of the reference item was 15 mg/L. The study met the acceptability criteria prescribed by the study plan and was considered valid.

The test item was shown to be not toxic to waste water bacteria (activated sludge) at a loading rate of 100 mg/L (30 min-NOEC). The 30min-EC50 was > 100 mg/L.

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