N-[4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino]phenyl]acetamide

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

no

Analytical monitoring:

not required

Details on sampling:

- Concentrations: 0.156, 0.3125, 0.625, 1.25, 2.5 mg/L

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test chemical for the required concentration was weighed and added to the medium.
- Controls:
Control blank: Prepred by adding test incoulum, synthetic sewage feed and water
Abiotic control: Prepared by addition of test chemical and synthetic sewage feed and water.
Positive control: Containing reference chemical (3,5-Dichlorophenol), inoculum, synthetic sewage feed and water
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): no vehicle was used

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Name and location of sewage treatment plant where inoculum was collected: The activated sludge was collected from SMS Municipal sewage treatment plant (130 MLD STP Bhandewadi, Nagpur, India) in a thoroughly cleansed container. The sampling site for collection of the activated sludge was selected ensuring that no known history of its contamination with the test item within the previous four years considering the history of possible agricultural, industrial or domestic inputs. The sampling depth was 1-2 feet from the aeration tank. The temperature of the activated sludge was measured (38º C) at the site of collection. Oxygen concentration of the activated sludge sample was 2.9 mg/L. The sample was transported to the test facility within 3 hours from collection and kept it aerobic during transport.

Method of cultivation: Aeration of incoming domestic sludge with micro-organisms by means of diffuser

Preparation of inoculum for exposure and pre-treatment:Sludge was pre-conditioned by decanting the supernatant of the activated sludge and later washed with sludge by mineral media followed by aerating for 1 day at the 20º C test temperature.
- Initial biomass concentration:

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Remarks on exposure duration:

All test chambers were aereated

Test temperature:

20°C

pH:

All vessel were having 7.4

Dissolved oxygen:

above 70% of ASV

Nominal and measured concentrations:

Nominal concentrations: 0.156, 0.3125, 0.625, 1.25, and 2.5 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Test vessel
- Type (delete if not applicable):loosly closed
- Material, size, headspace, fill volume: Glass 1L, heas space 600 ml and fill volume 400 ml
- Aeration: yes through out exposure duration (0.5 to 1L min)
- No. of vessels per concentration (replicates): 5 replicates per concentration
- No. of vessels per control (replicates): 6 vessels
- No. of vessels per vehicle control (replicates): no vehicle wasused
- No. of vessels per abiotic control (replicates): 6 abiotic control
- Sludge concentration (weight of dry solids per volume): 1.0 gram/L of suspended solids
- Weight of dry solids per volume of reaction mixture per unit of time:
- Nutrients provided for bacteria: synthetic sewage feed provided additional information
- Nitrification inhibitor used (delete if not applicable): N-allylthiourea
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: RO water.
OTHER TEST CONDITIONS
- Adjustment of pH: not adjusted

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2 factor
- Test concentrations: 0.156, 0.3125, 0.625, 1.25, and 2.5 mg/L

Reference substance (positive control):

yes

Remarks:

3,5 Dichlorophenol

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

0.15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

- Adsorption (e.g. of test material to the walls of the test container): No adsorption on the walls was observed
- Blank controls oxygen uptake rate: 34.3 mg/g/h
- Coefficient of variation of oxygen uptake rate in control replicates:13.4%

Results with reference substance (positive control):

- Results with reference substance valid? Valid
- Relevant effect levels: at concentration of 25 mg/L of 3,5 dichlorophenol, the inhibition was about 63.33%

Reported statistics and error estimates:

The percent inhibition was calculated using probit analysis using sys stat version 11.

Test Sample	Concentration of test chemical (mg/L)	pH	Respiration rate (mgO2/g/h)	% Inhibition
Control	0	-		0
Positive Control	25	7.4	12.5	63.33
Test Chemical	2.5	7.4	24	30.23
	1.25	7.4	24.9	27.62
	0.625	7.4	26.6	22.67
	0.3125	7.4	28.6	16.86
	0.156	7.4	33.4	2.91

O₂- Consumption

The oxygen consumption rate - R, in mg(O₂)/Lh, was calculated by measuring the decrease of oxygen content by Equation 1:

  R= (Q1 -Q2)*60/time                                                                                            (1)

Where;

Q1 is the oxygen concentration at the beginning of the incubation (mg/L);

Q2 is the oxygen concentration at the end of the incubation (mg/L).

 

2. Calculation of percentage of inhibition

The percentage inhibition, IT, of total oxygen consumption at each concentration of test substance, is given by Equation 2:

                                                                                    ITI= 100 - (RT*100)/Rc  (2)

 

Where;

ITI = inhibition of respiration (%)

RTI = O2 respiration rate of test item solutions [mg(O2)/Lh]

RC = O2 respiration rate of control (mean) [mg(O₂)/Lh]

 

3. Coefficient of Variation (%)

                                       Standard deviation *100/mean                                           (3)

Validity criteria fulfilled:

yes

Remarks:

% inhibition of 3,5-dichlorophenol was 63.33 mg/L at 25 mg/L Mean oxygen uptake rate was 1.14 Mean coefficient of variation was found to be 13.4% which is less than 30% The oxygen uptake rate of blank control was 34.3 mg/g/h which are > 20 mg/g/h.

Conclusions:

After the exposure duration of 3 hours the EC10 was reported to be 0.15 mg/L

Executive summary:

The microbial respiration study was conducted by following the OECD 209 guidelines, in which inhibition of respiration rate of inoculum was determined when exposed to test chemical for 3 hours. The activated sludge was collected from SMS Municipal sewage treatment plant (130 MLD STP) in a thoroughly cleansed container. The sampling site for collection of the activated sludge was selected ensuring that no known history of its contamination with the test item within the previous four years considering the history of possible domestic inputs. The sampling depth was 1-2 feet from the aeration tank. The temperature of the activated sludge was measured (38º C) at the site of collection. Oxygen concentration of the activated sludge sample was 2.9 mg/L. The sample was transported to the test facility within 1 hours from collection and kept it aerobic during transport.  Sludge was pre-conditioned by decanting the supernatant of the activated sludge and later washed by mineral media followed by aerating for 1 day at the 20º C test temperature. the test chemical and reference concentrations were prepared in 1000 ml glass beakers. The number of replicates in the control and abiotic control was 6 , and for test concentrations and reference compound the number of replicate were 5. the respective amount chemical was taken for preparing for concentrations ranging form 0.156 to 2.5 mg/l and reference compound of 25 mg/l were aerated (0.5 to 1 L/min) and incubated for a period of 3 hours at 20° C by adding a concentration of 32 mL/400 mL of activated sludge solution into every flask. This concentration gave suspended solid of 1.0 g/L. Later sample of each mixture was placed in a measuring (BOD) bottle (volume 125 mL) and the decrease in the oxygen concentration in each mixture was recorded with an oxygen electrode during the period of 5 minutes. The sensitivity of each batch of inoculum was checked with a positive control.

The effects on micro-organisms from inoculum were determined by measuring their respiration rate under defined conditions in the presence of different concentrations of the test substance. The respiration rates of samples of inoculum fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of three hours. Based outcomes on respiration inhibition the EC10 was reported to be 0.15 mg/L

Description of key information

Toxicity to microorganism:

The microbial respiration study was conducted by following the OECD 209 guidelines, in which inhibition of respiration rate of inoculum was determined when exposed to test chemical for 3 hours. The activated sludge was collected from SMS Municipal sewage treatment plant (130 MLD STP) in a thoroughly cleansed container. The sampling site for collection of the activated sludge was selected ensuring that no known history of its contamination with the test item within the previous four years considering the history of possible domestic inputs. The sampling depth was 1-2 feet from the aeration tank. The temperature of the activated sludge was measured (38º C) at the site of collection. Oxygen concentration of the activated sludge sample was 2.9 mg/L. The sample was transported to the test facility within 1 hours from collection and kept it aerobic during transport.  Sludge was pre-conditioned by decanting the supernatant of the activated sludge and later washed by mineral media followed by aerating for 1 day at the 20º C test temperature. the test chemical and reference concentrations were prepared in 1000 ml glass beakers. The number of replicates in the control and abiotic control was 6 , and for test concentrations and reference compound the number of replicate were 5. the respective amount chemical was taken for preparing for concentrations ranging form 0.156 to 2.5 mg/l and reference compound of 25 mg/l were aerated (0.5 to 1 L/min) and incubated for a period of 3 hours at 20° C by adding a concentration of 32 mL/400 mL of activated sludge solution into every flask. This concentration gave suspended solid of 1.0 g/L. Later sample of each mixture was placed in a measuring (BOD) bottle (volume 125 mL) and the decrease in the oxygen concentration in each mixture was recorded with an oxygen electrode during the period of 5 minutes. The sensitivity of each batch of inoculum was checked with a positive control.

The effects on micro-organisms from inoculum were determined by measuring their respiration rate under defined conditions in the presence of different concentrations of the test substance. The respiration rates of samples of inoculum fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of three hours. Based outcomes on respiration inhibition the EC10 was reported to be 0.15 mg/L

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

0.15 mg/L

Additional information

Toxicity to microorganism:

The microbial respiration study was conducted by following the OECD 209 guidelines, in which inhibition of respiration rate of inoculum was determined when exposed to test chemical for 3 hours. The activated sludge was collected from SMS Municipal sewage treatment plant (130 MLD STP) in a thoroughly cleansed container. The sampling site for collection of the activated sludge was selected ensuring that no known history of its contamination with the test item within the previous four years considering the history of possible domestic inputs. The sampling depth was 1-2 feet from the aeration tank. The temperature of the activated sludge was measured (38º C) at the site of collection. Oxygen concentration of the activated sludge sample was 2.9 mg/L. The sample was transported to the test facility within 1 hours from collection and kept it aerobic during transport.  Sludge was pre-conditioned by decanting the supernatant of the activated sludge and later washed by mineral media followed by aerating for 1 day at the 20º C test temperature. the test chemical and reference concentrations were prepared in 1000 ml glass beakers. The number of replicates in the control and abiotic control was 6 , and for test concentrations and reference compound the number of replicate were 5. the respective amount chemical was taken for preparing for concentrations ranging form 0.156 to 2.5 mg/l and reference compound of 25 mg/l were aerated (0.5 to 1 L/min) and incubated for a period of 3 hours at 20° C by adding a concentration of 32 mL/400 mL of activated sludge solution into every flask. This concentration gave suspended solid of 1.0 g/L. Later sample of each mixture was placed in a measuring (BOD) bottle (volume 125 mL) and the decrease in the oxygen concentration in each mixture was recorded with an oxygen electrode during the period of 5 minutes. The sensitivity of each batch of inoculum was checked with a positive control.

The effects on micro-organisms from inoculum were determined by measuring their respiration rate under defined conditions in the presence of different concentrations of the test substance. The respiration rates of samples of inoculum fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of three hours. Based outcomes on respiration inhibition the EC10 was reported to be 0.15 mg/L

 

The test material was evaluated for its antimicrobial effect on Tetrahymena pyriformis. The inhibitory effect concentration (IC50) for Tetrahymena pyriformis was observed to be 0.5 mg/l . Nonetheless, the test material also inhibited the protozoan reproduction.

 

For the test chemical, another short term toxicity to Vibrio fischeri study was carried out.The study was performed in accordance with the standard method ‘DIN 38412 Part 34’. On the basis of the effect of test chemical on luminescence of the test organism Vibrio fischeri, the EC20 value was determined to be 0.5 mg/l.

 

On the basis of the experimental studies of the structurally and functionally similar read across chemical and applying the weight of evidence approach, both the IC50 and EC20 value the test chemical on the test organism was observed to be 0.5 mg/l.