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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
August 2019 - October 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
OECD (2010)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Chemical name: Substituted amino acid (2) solution FC-C 13587
Assay: 27.8% active; 72.6% water
Batch/lot No.: FC-C 13587
Appearance: Clear colorless solution
Solubility: Soluble in water at test concentrations
Volatile: Expected non-volatile
Expiry date
March 2020 (if stored at max -18°C)
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Activated sludge
Unadapted secondary activated sludge was obtained (28-08-2019) from the WWTP Nieuwgraaf in Duiven. The WWTP Duiven is an activated sludge plant treating predominantly domestic wastewater. Prior to use the activated sludge was homogenized with a syringe. The measured dry weight of the homogenized activated sludge was 3.4 g/L and was concentrated by settlement to 6.0 g/L dry weight. Subsequently, the concentrated activated sludge was diluted in the incubation vessels to an activated sludge dry weight concentration of 1.5 g/L.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Remarks on exposure duration:
max value according to Guideline
Test temperature:
The temperature of the BOM thermostated vessel measured at the start, middle and end of the test varied from 19.1 to 19.6 °C. The temperature of the water bath, used for the incubation of the activated sludge, at the beginning, middle and end of the test was 20.2 °C, 20.2 °C and 20.3 °C, respectively. Temperatures measured in the Erlenmeyer flasks at the end of the incubation period varied from 20.0 to 20.4 °C.
pH:
The pH of the reaction mixtures after the incubation period ranged from 7.0 to 7.3
Dissolved oxygen:
is measured
Salinity:
Freshwater
The synthetic sewage feed was made by dissolving the following amounts of substances in 1 liter of deionized water: 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. In the incubation vessels 1.6 mL and 6.4 mL of the synthetic sewage feed is added in a total volume of 50 and 200 mL, respectively.
Details on test conditions:
Test procedures
The activated sludge respiration inhibition test was performed according to the study plan. The study plan was developed from an OECD Test Guideline (OECD, 2010). In this test the inhibition of oxygen uptake by micro-organisms oxidizing organic carbon was not separately expressed from the respiration by micro-organisms oxidizing ammonium. The total respiration rate of activated sludge fed with a standard amount of synthetic sewage was measured. The respiration rate of the same activated sludge in the presence of various concentrations of the test substance under otherwise identical conditions was also measured. The inhibitory effect of the test substance at a particular concentration was expressed as a percentage of the mean respiration rates of the controls. Some aspects of the test are listed below:
• Prior to use the activated sludge was homogenized with a syringe.
• The activated sludge with test substances was incubated in a shaking bath (100 rpm/min) for 3 hours.
• The test substance and the reference compound (3,5-dichlorophenol) concentrations were spaced by a factor two.
• An abiotic control was not performed because neither reduction of oxygen by the test substance nor formation of oxygen due to chemical breakdown of the test substance is expected.
• No specific analyses were performed.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol (p.a. quality 99.1%) used as a reference substance in the activated sludge respiration inhibition test was purchased from Sigma Aldrich.
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
591 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95% CL: 276 - 2933 mg/L
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
164.3 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
Remarks on result:
other: 95% CL: 76.7 - 815.4 mg/L
Details on results:
Test conditions
The pH of the reaction mixtures after the incubation period ranged from 7.0 to 7.3. The temperature of the BOM thermostated vessel measured at the start, middle and end of the test varied from 19.1 to 19.6 °C. The temperature of the water bath, used for the incubation of the activated sludge, at the beginning, middle and end of the test was 20.2 °C, 20.2 °C and 20.3 °C, respectively. Temperatures measured in the Erlenmeyer flasks at the end of the incubation period varied from 20.0 to 20.4 °C. These conditions allow respiration of the activated sludge used.

Validity of the test
The validity of the test is demonstrated although only two out of three validity criteria were met according the guideline. First, the prescribed oxygen uptake rate of >20 mg O2/g dry weight/hour for the control measurements was not met. The inoculum used was obtained from a treatment plant which is operated at a low organic sludge load and as a consequence has a low oxygen uptake rate. Operating at low organic sludge loads is nowadays very common. The average oxygen uptake rate for the control replicates was 12 mg O2/g dry weight/hour, which is lower than the prescribed rate. This does not influence the sensitivity of the activated sludge as shown by comparable EC50 values of the reference substance 3,5-dichlorophenol at oxygen uptake rates lower and higher than the prescribed rate (AkzoNobel 2012). The lower average oxygen uptake rate does therefore not compromise the test outcome in terms of robustness of the endpoints obtained for the test and reference substance and does not invalidate the test. Second, the coefficient of variation of the replicates of the control oxygen uptake rates was 17%, which shows a high precision and fulfills the maximum prescribed variation of <30%.
Third, the EC50 of the reference compound for activated sludge after 3 hours contact time is 8 mg/L, which is within the prescribed range of 2 to 25 mg/L (Table I and Figure 1).

Activated sludge respiration inhibition test
The calculated respiration rates and inhibition percentages of substituted amino acid (2) solution FC-C 13587 are presented in Table II and the results of the graphical probit analysis are shown in Figure 2. The inhibitory effect of substituted amino acid (2) solution FC-C 13587 at a particular concentration is expressed as a percentage of the two controls. From the results EC values were calculated. The EC10 of substituted amino acid (2) solution FC-C 13587 for activated sludge after 3 hours contact time is 591 mg/L with 95% confidence limits of 276 and 2933 mg/L. The EC20, EC50 and EC80 values could not be determined. Substituted amino acid (2) solution FC-C 13587 is therefore considered not harmful to activated sludge.
Results with reference substance (positive control):
The EC50 of the reference compound for activated sludge after 3 hours contact time is 8 mg/L, which is within the prescribed range of 2 to 25 mg/L
Reported statistics and error estimates:
ToxRat Solutions GmbH 2001-2010. Toxicity Response Analysis and Testing Software version 2.10

Table I Respiration rates of the activated sludge, inhibition percentages, pH and temperature at various concentrations of 3,5-dichlorophenol after 3 hours contact time.

Concentration

(mg/L)

Activity

(mg O2/g/min)

Inhibition (%)

pH at start test

pH at end test

Temperature at end test

(°C)

Control

0.2667

-

7.1

7.2

20.2

Control

0.1944

-

7.0

7.2

20.2

Control*

0.1905

-

7.1

7.2

20.2

Control*

0.1852

-

7.1

7.2

20.3

1.27*

0.1667

11

7.1

7.2

20.2

2.54

0.1481

36

7.1

7.2

20.2

5.09

0.1567

32

7.1

7.2

20.1

10.18

0.1037

55

7.1

7.2

20.1

20.36

0.0630

73

7.0

7.2

20.2

40.71

0.0333

86

7.0

7.3

20.0

pH stock solution 3,5-dichlorophenol = 6.7

pH synthetic sewage = 7.1

* = corresponding control and reference concentration

 

 

Table II Respiration rates of the activated sludge, inhibition percentages, pH and temperature at various concentrations of sodium hydrogen substituted amino acid (2) solution FC-C 13587 after 3 hours contact time.

Concentration

(mg/L)

Activity

(mg O2/g/min)

Inhibition (%)

pH at start test

pH at end test

Temperature end test

(°C)

Control

0.1833

-

7.1

7.2

20.3

Control

0.1905

-

7.1

7.2

20.2

Control*

0.1905

-

7.1

7.2

20.2

Control*

0.1852

-

7.1

7.2

20.3

20.0

0.2083

-11

7.2

7.2

20.3

40.0

0.1917

-3

7.2

7.2

20.2

80.0

0.2083

-11

7.2

7.2

20.2

160.0

0.1963

-5

7.2

7.2

20.4

319.9

0.2000

-7

7.2

7.2

20.4

639.9#*

0.1667

11

6.9

7.1

20.3

1279.9#*

0.1556

17

6.7

7.0

20.2

Concentration stock solution sodium hydrogen substituted amino acid (2) solution FC-C 13587 = 1 g/L, pH stocksolution used = 3.5

# Concentration stock solution sodium hydrogen substituted amino acid (2) solution

FC-C 13587 = 2 g/L, pH stocksolution used = 3.4

* = corresponding control and test substance concentration

Validity criteria fulfilled:
yes
Conclusions:
The test is valid as shown by the EC50 of the reference compound 3,5-dichlorophenol (8 mg/L) and the coefficient of variation of the replicates of the control oxygen uptake rates (16%). The average oxygen uptake rate for the control replicates is lower (13 mg O2/g dry weight/hour) than the prescribed rate (>20 mg O2/g dry weight /hour). This does not influence the sensitivity of the activated sludge as shown by comparable EC50 values of the reference substance 3,5-dichlorophenol at oxygen uptake rates lower and higher than the prescribed rate (AkzoNobel 2012). The lower average oxygen uptake rate does therefore not compromise the test outcome in terms of robustness of the endpoints obtained for the test and reference substance and does not invalidate the test.
Executive summary:

In order to predict effects of chemicals in the environment and in biological waste water treatment plants, an activated sludge respiration inhibition test was performed. The toxicity to activated sludge was determined in accordance with OECD Test Guideline 209, and in compliance with the OECD principles of Good Laboratory Practice.

The toxicity of substituted amino acid (2) solution FC-C 13587 to activated sludge was determined at a contact time of 3 hours, using various concentrations of the test substance. The inhibitory effect of substituted amino acid (2) solution FC-C 13587 at a particular concentration is expressed as a percentage of the two controls. From the results EC values were calculated. The EC10 of substituted amino acid (2) solution FC-C 13587 for activated sludge after 3 hours contact time is 591 mg/L with 95% confidence limits of 276 and 2933 mg/L. The EC20, EC50 and EC80 of substituted amino acid (2) solution FC-C 13587 for activated sludge could not be determined. Substituted amino acid (2) solution FC-C 13587 is therefore considered not harmful to activated sludge.

Description of key information

The ecotoxicity data from Substituted amino acid (2) solution is read across to Sodium salts of substituted amino acid (2). This read across is considered justified as during ecotoxicity testing, diluted test concentrations are prepared in a matrix with sufficient buffer capacity. At higher concentrations e.g. in stock solutions they are neutralized when the pH is outside the biologically optimal range. The difference between both substances is therefore considered negligible in the perspective of ecotoxicity testing.

The toxicity of substituted amino acid (2) solution FC-C 13587 to activated sludge was determined according to OECD 209 under GLP conditions at a contact time of 3 hours, using various concentrations of the test substance. The inhibitory effect of substituted amino acid (2) solution FC-C 13587 at a particular concentration is expressed as a percentage of the two controls. From the results EC values were calculated. The EC10 of substituted amino acid (2) solution FC-C 13587 for activated sludge after 3 hours contact time is 164.3 mg a.i./L with 95% confidence limits of 76.7 and 815.4 mg/L. The EC20, EC50 and EC80 of substituted amino acid (2) solution FC-C 13587 for activated sludge could not be determined. Substituted amino acid (2) solution FC-C 13587 is therefore considered not harmful to activated sludge.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
164.3 mg/L

Additional information

Good quality data obtained with other types of microbial inhibition test methods, degradation- or sewage treatment simulation tests, can be also used to meet the REACH requirements