[No public or meaningful name is available]

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 February to 20 March 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline 209. All validity criteria were fulfilled

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Specific details on test material used for the study:

- Physical state: Colorless liquid
- Storage condition of test material: Stored in refrigerator (2-8°C) protected from light, container flushed with nitrogen
- Others:
Volatile: Yes, vapour pressure: 1.51 Pa
Specific gravity/density: 0.87 g/mL (determined by WIL Research Europe)
Solubility in water: 7.25 mg/L
Stability in water: Stable

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test substance was not sufficiently soluble to allow the preparation of a 10 g/L stock solution in water. Therefore, 1-Litre test bottles were filled with 200 mL of test substance mixtures in Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with initial loading rates of 2.5 times the final loading rate. The test substance was pipetted under the medium surface in the individual vessels. These mixtures were stirred in closed dark brown bottles for 5 to 13 minutes. Because of the volatile characteristics of the test substance the solutions were not stirred for 24 hours. Subsequently, 16 mL synthetic medium made up to 50 mL with Milli-RO water and 250 mL sludge were added resulting in the required loading rates. Optimal contact between the test substance and test organisms was ensured applying continuous aeration and stirring.
- Controls: Test medium without test substance and treated in the same way as the test substance solutions.

Test organisms (species):

other: Micro-organisms in activated sludge

Details on inoculum:

- Source: Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Preparation of the sludge: 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). The pH was 5.7 and adjusted to 7.3 using 1M NaOH (Merck, Darmstadt, Germany) on the day of testing. The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.

Test type:

not specified

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

None

Hardness:

None

Test temperature:

18-22 °C

pH:

The pH in all test vessels, before addition of sludge was between 7.4 and 7.7. After the 3 hour exposure period the pH was between 6.4 and 7.5.

Dissolved oxygen:

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.

Salinity:

None

Nominal and measured concentrations:

Five loading rates: 46, 100, 220, 460 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: All glass open bottles/vessels
- Aeration: 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.
- Air supply: Clean, oil-free air
- No. of vessels per concentration (replicates): 5 replicates per test group
- No. of vessels per control (replicates): 6 replicates for the control

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO / Milli-RO water (Tap-water purified by reverse osmosis (Millipore Corp., Bedford, Mass., USA))
- Medium: Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition: CaCl2.2H2O: 211.5 mg/L; MgSO4.7H2O: 88.8 mg/L; NaHCO3: 46.7 mg/L; KCl: 4.2 mg/L
- Synthetic medium (=sewage feed): 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 Milli-RO water, made up to 1 litre and filtered. The pH was within 7.5 ± 0.5.

PERFORMANCE OF THE TEST
- The synthetic medium (16 mL) made up to 50 mL with Milli-RO and 200 mL test substance solution were mixed (total volume 250 mL) in a 1 litre bottle. The pH was determined. Thereafter 250 mL activated sludge was added. This was the start of the test. After the 3-hour contact time, the oxygen consumption was recorded for a period of approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer.
- The pH was determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference substance concentrations and controls. The medium temperature was recorded continuously in a temperature control vessel(s). The temperature control vessel(s) was/were identically prepared compared to the control vessels. A temperature control vessel with a REES sensor was placed in each fume cupboard of the climate room.
- Oxygen recording: Determination of oxygen was performed with multiple oxygen probes connected to a BlueBox (GO-Systemelektronik GmbH, Germany), a multichannel measuring and controlling system.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Range finding study: In a combined limit/range-finding test loading rates of 10, 100 and 1000 mg/Lwere tested. The highest loading rate was tested in triplicate, lower loading rates consisted of one replicate. In addition, a blank control (6 replicates) and a nitrification control (2 replicates) were included. Furthermore, an abiotic control (1 replicate) and the highest loading rate with a nitrification inhibitor (3 replicates) were tested.
Test procedure and conditions were similar to those applied in the final test with the following exceptions:
- The pH of the sludge was 7.1 on the day of testing.
- Inhibitor of nitrification: A 2.32 g/L solution of N-allylthiourea (ATU, Merck Schuchardt OHG, Hohenbrunn, Germany) was prepared. 2.5 mL of this solution was added to 500 mL final test medium (final ATU concentration: 11.6 mg/L).
- Results used to determine the conditions for the definitive study:
The combined limit/range-finding test showed no biological relevant inhibition at 10 and 100 mg/L and almost complete inhibition at a loading rate of 1000 mg/L.
There was no oxygen uptake from abiotic processes and the result at 1000 mg/L with a nitrification inhibitor showed that the heterotrophic inhibition of the respiration rate was comparable to the total inhibition. Based on the results of the total and heterotrophic respiration, nitrification inhibition was calculated to be also completely inhibited.
The temperature continuously measured in the temperature control vessels ranged between 19 and 21 °C during the test, and complied with the requirements as laid down in the protocol (20 ± 2°C).
The pH in the controls, before addition of sludge was 7.9. After the 3 hour exposure period the pH was between 7.2 and 8.0. The pH in the test vessels, before addition of sludge was between 5.6 and 5.9. After the 3 hour exposure period the pH was between 7.4 and 7.6.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol at 2.0, 5.0 and 12 mg/L

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

<= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

130 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: 95% confidence interval: 110 – 140 mg/L

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

250 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: 95% confidence interval: 240 – 270 mg/L

Details on results:

Inhibition of the respiration rate: No statistically significant inhibition of the respiration rate of the sludge was recorded at or below a loading rate of 100 mg/L. At higher loading rates the inhibitory effect of test substance on aerobic waste water (activated sludge) bacteria increased with increasing loading rate, ranging from 39% inhibition at 220 mg/L to 98% at 1000 mg/L.

Results with reference substance (positive control):

The batch of activated sludge was tested for sensitivity with the reference substance 3,5-dichlorophenol, and showed normal sensitivity. EC50 value of 3,5-dichlorophenol was 4.43 mg/L.

Reported statistics and error estimates:

ECx and ELx: For the reference substance calculations calculation of EC50 values was based on probit analysis using linear maximum likelihood regression with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the test substance.
Calculation of ELx values were based on probit analysis using linear maximum likelihood regression with the percentages of respiration inhibition versus the logarithms of the corresponding loading rates of the test substance
NOEL determination: An effect was considered to be significant if statistical analysis of the data obtained for the test loading rates compared with those obtained in the blank control revealed significant inhibition of the respiration rate (student-t test for Homogeneous Variances with Bonferroni-Holm Adjustment, α=0.05, one-sided, smaller).
The calculations were performed with ToxRat Professional v. 2.10.05 (ToxRat Solutions® GmbH, Germany).

Table 6.1.7/3: Final test – Overview of the results

 

Treatment	Loading rate (mg/L)	pH		Mean respiration rate		% Inhibition of the respiration rate (mean value)
		Start	End	(mg O2/L h)	(mg O2/g h)^1
Blank control	-	7.6-7.7	6.4-6.7	39	26	-
T1	46	7.4	6.4-6.6	41	27	-6
T2	100	7.6	6.6-6.7	36	24	6
T3	220	7.6	6.9-7.1	24	16	39*
T4	460	7.6	6.7-6.9	5	3	87*
T5	1000	7.6	6.8	1	0	98*

¹)The amount of suspended solids in the final test mixture was 1.5 g/L.

* Statistically significantly different compared to control

 

Acceptability of the test

The mean blank control oxygen uptake rate exceeded 20 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour (24 and 26 mg oxygen per one gram of activated sludge, for the combined limit/range-finding and final test respectively). 

The coefficient of variation of oxygen uptake in blank control replicates did not exceed 30% (12% in both tests).

The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/L for total respiration (5.1 and 4.4 mg/L, for the combined limit/range-finding and final test respectively.

Since all criteria for acceptability of the test were met, this study was considered to be valid.

Validity criteria fulfilled:

yes

Conclusions:

Under the test conditions, the test substance was not toxic to waste water bacteria (activated sludge) at or below a loading rate of 100 mg/L (NOEL). The EL10 was at a loading rate of 130 mg/L (95% confidence interval: 110 – 140 mg/L). The EL50 was at a loading rate of 250 mg/L (95% confidence interval: 240 – 270 mg/L).

Executive summary:

The effect of test substance on the respiration rate of activated sludge was investigated after a contact time of 3 h, according to OECD Guideline 209 with GLP statement.

The test substance was pipetted under the medium surface in the individual vessels. The test substance – Milli-RO water mixtures were magnetically stirred for a period of 5 to 13 minutes. Subsequently, synthetic medium, sludge and Milli-RO water were added resulting in the required loading rates. Optimal contact between the test substance and test medium was ensured applying continuous aeration and stirring during the 3-hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes.

The final test was performed based on the result of a preceding combined limit/range-finding test. Five loading rates, were employed at 46, 100, 220, 460 and 1000 mg/L, increasing with a factor 2.2 were tested together with a control. Five replicates per loading rate and six replicates for an untreated control group were tested.

 

No statistically significant inhibition of the respiration rate of the sludge was recorded at or below a loading rate of 100 mg/L. At higher loading rates the inhibitory effect of test substance on aerobic waste water (activated sludge) bacteria increased with increasing loading rate, ranging from 39% inhibition at 220 mg/L to 98% at 1000 mg/L.

 

The batch of activated sludge was tested for sensitivity with the reference substance 3,5-dichlorophenol, and showed normal sensitivity.

 

The study met the acceptability criteria prescribed by the protocol and was considered valid.

Under the test conditions, the test substance was not toxic to waste water bacteria (activated sludge) at or below a loading rate of 100 mg/L (NOEL). The EL10 was at a loading rate of 130 mg/L (95% confidence interval: 110 – 140 mg/L). The EL50 was at a loading rate of 250 mg/L (95% confidence interval: 240 – 270 mg/L).

Description of key information

OECD Guideline 209, GLP, key study, validity 1

3h-EC50 (activated sludge of a municipal sewage treatment plant) =250 mg/L based on nominal concentration

3h-EC10 (activated sludge of a municipal sewage treatment plant) = 130 mg/L based on nominal concentration

3h-NOEC (activated sludge of a municipal sewage treatment plant) = 100 mg/L based on nominal concentration

Key value for chemical safety assessment

EC50 for microorganisms:

250 mg/L

EC10 or NOEC for microorganisms:

100 mg/L

Additional information

One key study is available (WIL, 2015) 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 h, according to OECD Guideline 209 with GLP statement.

The registered substance was pipetted under the medium surface in the individual vessels. The test substance – Milli-RO water mixtures were magnetically stirred for a period of 5 to 13 minutes. Subsequently, synthetic medium, sludge and Milli-RO water were added resulting in the required loading rates. Optimal contact between the test substance and test medium was ensured applying continuous aeration and stirring during the 3-hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes.

The final test was performed based on the result of a preceding combined limit/range-finding test. Five loading rates, ranging from 46, 100, 220, 460 and 1000 mg/L, increasing with a factor 2.2 were tested. Five replicates per loading rate and six replicates for an untreated control group were tested.  The inhibitory effects of the test and reference substances were determined in comparison to the control respiration rates. No statistically significant inhibition of the respiration rate of the sludge was recorded at or below a loading rate of 100 mg/L. At higher loading rates the inhibitory effect of test substance on aerobic waste water (activated sludge) bacteria increased with increasing loading rate, ranging from 39% inhibition at 220 mg/L to 98% at 1000 mg/L.The batch of activated sludge was tested for sensitivity with the reference substance 3,5-dichlorophenol, and showed normal sensitivity. The study met the acceptability criteria prescribed by the protocol and was considered valid.

Under the test conditions, the registered substance was not toxic to waste water bacteria (activated sludge) at or below a loading rate of 100 mg/L (NOEL). The 3h-EL10 was at a loading rate of 130 mg/L (95% confidence interval: 110 – 140 mg/L). The 3h-EL50 was at a loading rate of 250 mg/L (95% confidence interval: 240 – 270 mg/L).