2-methyl-p-phenylenediamine sulfate

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-05-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and Guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-methyl-p-phenylenediamine sulfate

EC Number:

210-431-8

EC Name:

2-methyl-p-phenylenediamine sulfate

Cas Number:

615-50-9

Molecular formula:

C7H10N2.H2O4S

IUPAC Name:

2-methylbenzene-1,4-diamine; sulfuric acid

Constituent 2

Reference substance name:

2-methyl-p-phenylenediamine sulphate

EC Number:

228-871-4

EC Name:

2-methyl-p-phenylenediamine sulphate

Cas Number:

6369-59-1

IUPAC Name:

2-methylbenzene-1,4-diamine sulfate

Details on test material:

- Name of test material): 2-methyl-p-phenylenediamine sulfate (Haarbraun)
- Substance type: Pure active substance
- Physical state: White to light pink powder

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Direct weighing of test material into stock solution flask. Stock solution of 2000 mg/L was prepared.
- Controls: 2 control samples without test item were included in every test series.
- Chemical name of vehicle: not used
- Evidence of undissolved material: none

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Source of inoculum/activated sludge: Non-adapted activated sludge from the sewage plant at D-31157 Sarstedt, collected the day before testing.
- Laboratory culture: N/A
- Storage conditions: N/A
- Pretreatment: The sludge was washed with autoclaved tap water and diluted 4.0 g/L (dry weight, +/- 10%).
- Concentration of sludge: 3.60 g/L dry weight

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

None

Test conditions

Hardness:

N/A

Test temperature:

22 degrees Celsius

pH:

6.88

Dissolved oxygen:

N/A

Salinity:

N/A

Nominal and measured concentrations:

Nominal - 0 (control), 1.0, 3.20, 10.0, 32.0, and 100.0 mg/L; Dilution factor 3.2
Reference substance concentrations: 58, 100, 180 mg/L; Dilution factor 1.8

Details on test conditions:

TEST CONDITIONS
- Volume of test solution/treatment: 500 ml
- Composition of medium: The test medium was comprised of the inoculum (activated sludge), synthetic waste water, autoclaved tap water, and the test material. For example, the composition of the 1.0 mg/L test concentration was as follows:
200 ml inoculum (activated sludge)
16 ml synthetic waste water (prepared according to OECD 209)
283.75 ml autoclaved tap water
0.25 ml stock solution of test item
500 ml Total
- Test temperature: 22 degrees C
- pH: 6.88
- pH adjusted: no
- Aeration of dilution water: N/A
- Suspended solids concentration: In the test, the concentration of solids was 1.6 g/L (dry weight).
- Continuous darkness: no
- Any indication of the test material adsorbing to the walls of the test apparatus: No
- Other: Copper (ll) sulphate pentahydrate reference substance used; Source- Merck


TEST SYSTEM
- Culturing apparatus: 500 ml Erlenmeyer flask
- Number of culture flasks/concentration: 1 replicate per test concentration, two per control
- Method used to create aerobic conditions: the test medium was saturated with oxygen prior to start of the test.
- Measuring equipment: Oximeter, WTW, Oxi 197-S was used to record the oxygen depletion after 3 hour incubation
- Test performed in closed vessels due to parameter measured: yes
- Other: 5 concentrations within the range of 1-100 mg/L, in a geometrical series with a dilution factor of 3.2 were tested; 2 controls without test substance were also included.

SAMPLING
- Sampling frequency: Oxygen depletion was measured after 3 hr (at completion of test).


CONTROL AND BLANK SYSTEM
- Inoculum blank: no
- Abiotic sterile control: no
- Toxicity control: no

Reference substance (positive control):

yes

Remarks:

Copper (II) sulphate pentahydrate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

8.4 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Effect concentrations 2

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

17.7 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% CI is 15.3-20.3 mg/L

Effect concentrations 3

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

37 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

Inhibition ranged from 3 to 84% in the concentration range of 3.2 mg/l to 100 mg/l. The calculated EC50 was 17.7 mg/L (15.3 - 20.3 mg/L).

Results with reference substance (positive control):

The EC50 for the reference substance was 120 mg/L, with a 95% confidence interval of 53-155 mg/L. This is within acceptable limits.

Reported statistics and error estimates:

EC values were calculated with probit analysis using SIGMAPLOT software 6.0

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

EC50 of the reference substance was 120 mg/L, which is within required limits of 53-155 mg/L, and the difference between the controls is 3%, which is within the allowable maximum of 15%. Therefore, the test is valid in accordance with Guideline 209.

Conclusions:

In a 3 hr activated sludge respiration inhibition test, the EC50 of 2-methyl-p-phenylenediamine sulfate was 17.7 mg/L (95% CI of 15.3-20.3 mg/L).

Executive summary:

An activated sludge respiration inhibition test of 2 -methyl-p-phenylenediamine sulfate was conducted according to the OECD 209 Guideline. Nominal test concentrations were 0 (control), 1.0, 3.20, 10.0, 32.0, and 100 mg/L. The reference substance was Copper (II) sulphate pentahydrate. The respiration rates of the control, reference, and test substance concentrations were measured after a contact time of three hours, and the inhibitory effects of the test and reference concentrations were determined in comparison to the control respiration rates. The inhibition ranged from 3 to 84 %. The EC20 was 8.4 mg/L, the EC50 was 17.7 (95% CI 15.3 -20.3 mg/L), and the EC80 was 37.0 mg/L. The EC50 for the reference substance was 120 mg/L, which was within acceptable limits. The test met all the validity criteria for the OECD 209 test.