4,4'-benzylidenedianiline

Toxicity to microorganisms

Administrative data

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:

guideline study

Remarks:

Study based on older version of OECD Guideline (concurrent version at the time of the study)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

April 4th 1984 version

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-benzylidenedianiline

EC Number:

210-038-1

EC Name:

4,4'-benzylidenedianiline

Cas Number:

603-40-7

Molecular formula:

C19H18N2

IUPAC Name:

4-[(4-aminophenyl)(phenyl)methyl]aniline

Test material form:

solid

Details on test material:

Identification: DATIPH
Physical form: Solid
Solubility in water: < 1 g/L
Colour: yellow brown
CAS no.: 603-40-7
Chemical name: 4,4’-Benzylidenedianiline
Molecular formula: C19H18N2
Test substance storage: At room temperature protected from light
Stability: Stable under storage conditions
Expiry date: 31 October 2010

Specific details on test material used for the study:

Identification: DATIPH
Chemical name: 4,4’-Benzylidenedianiline
Physical form: Solid
Colour: Yellow brown
Solubility in water: < 1 g/L (virtually insoluble)
CAS no.: 603-40-7
EINECS no.: 210-038-1
Molecular formula: C19H18N2
Test substance storage: At room temperature, protected from light
Stability: Stable under storage conditions
Expiry date: 31.10.2010

Sampling and analysis

Analytical monitoring:

no

Remarks:

No analysis of test concentrations was conducted. For the estimation of the EC20, EC50 and EC80 values the nominal content of the test substance in the test media was used.

Details on sampling:

A range finding test with 10, 100, and 1000 mg/l nominal concentrations was conducted.
Final test concentrations were prepared by directly adding the respective amount of the test substance to the test vessels.

Test solutions

Vehicle:

yes

Remarks:

Test medium

Details on test solutions:

A range finding test with 10, 100, and 1000 mg/l nominal concentrations was conducted. Since no toxic effects were observed this report is based on the result of this screening test.
Final test concentrations were prepared by directly adding the respective amount of the test substance to the test vessels.
For each test series the following number of test flasks was set up:
• Test suspension (Tn); one replicate sample containing activated sludge + test medium + test substance
• Blank control (B1, B2); two replicate samples containing activated sludge + test medium
• Sensitivity control (Rn); one replicate sample (3 test concentrations) containing activated sludge + test medium + 3,5-dichlorophenol as sensitivity control

Test organisms

Test organisms (species):

activated sludge

Details on inoculum:

Not adapted and not pre-conditioned activated sludge from the aeration tank of the ARA Werdhòlzli (CH-8048 Zürich), a municipal biological waste water treatment plant. 1.6 g/l dry matter in the final mixture.

Study design

Test type:

static

Water media type:

other: Tap water + synthetic sewage feed

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

None.

Test conditions

Hardness:

Between 36.6 and 37.5 °F

Test temperature:

22 ± 2 °C

pH:

All values were within an acceptable range.
Tap water pH values are between 7.2 and 7.4.

Dissolved oxygen:

Not specified

Salinity:

As given by the composition of the test medium (see table below).

Conductivity:

Tap water conductivity is between 691 and 731 µS/cm

Nominal and measured concentrations:

Nominal concentrations = 10, 100 and 1000 mg/L.
No measure of the test substance concentration was carried out.

Details on test conditions:

TEST SYSTEM AND TEST CONDITIONS:
Inoculum: Not adapted and not pre-conditioned activated sludge from the aeration tank of the ARA Werdhòlzli (CH-8048 Zürich), a municipal biological waste water treatment plant. 1.6 g/l dry matter in the final mixture.
Sampling: 24 November 2009; 9.15 a.m.
Test units: 1000 ml flasks containing a total volume of test suspension of 500 ml; aerated by shaking (120 r.p.m.).
Measuring apparatus: 300 ml flat bottom BOD flasks; polarographic oxygen electrode, connected to a potentio-metric recorder.
Duration/contact time: 3 hours in a temperature-controlled room.
Temperature: 22 ± 2 °C.
Test medium: Synthetic sewage feed (Table 1) prepared with tap water (Table 2): 16 ml of 100-fold sewage concentrate per 500 ml of final test mixture.
Sensitivity control: 3,5-Dichlorophenol.

TEST PROCEDURE:
Since the activated sludge was used two days after the sludge sampling, 50 ml of the synthetic sewage feed (Table 1) were added per litre of activated sludge and the sludge was continuously aerated. Prior to the test, the sludge was washed twice with tap water (Table 2).
The test vessels contained activated sludge in an amount corresponding to 1.6 g/l dry matter in the final mixture.
A range finding test with 10, 100, and 1000 mg/l nominal concentrations was conducted. Since no toxic effects were observed this report is based on the result of this screening test.
Final test concentrations were prepared by directly adding the respective amount of the test substance to the test vessels.
For each test series the following number of test flasks was set up:
• Test suspension (Tn); one replicate sample containing activated sludge + test medium + test substance
• Blank control (B1, B2); two replicate samples containing activated sludge + test medium
• Sensitivity control (Rn); one replicate sample (3 test concentrations) containing activated sludge + test medium + 3,5-dichlorophenol as sensitivity control
The test vessels were aerated by shaking (120 r.p.m.) for the whole test period of 3 hours. The first sample (blank control B1 was analyzed 3 h after starting the test
followed by the analysis of the sensitivity controls (Rn) and the test suspensions (Tn) at 5-15 min intervals. Finally, the second blank control B2 was analyzed.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol.

Results and discussion

Effect concentrations

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

inhibition of total respiration

Details on results:

At all applied test concentrations (10, 100 and 1000 mg/L), the test substance showed no significant toxic effects to the activated sludge. The respiration rates were in the same range as determined for the two blank controls.

Results with reference substance (positive control):

The EC50 value of the reference substance 3,5-dichlorophenol was determined to be 4.37 mg/l (95% confidence limits: n.d.-13.2 mg/l) which is in the 2-25 mg/L range for total respiration measurements. Hence, the suitability of activated sludge and test conditions was shown. The variation between the two controls was ± 2.0%. Therefore, the test was considered valid.

Any other information on results incl. tables

Table 1: Activated sludge respiration rates after 3 h of exposure to the test material and calculation of the inhibition

Nominal concentration (mg/l)		Code	O2consumption rates (mg O2*l^-1*h^-1)	Respiration inhibition (%)
Control	0	B1	29.9	-
	0	B2	31.1
10		T1	32.0	-5.1
100		T2	30.8	-1.2
1000		T3	31.3	-2.7
Reference	3	R1	17.9	41.2
	10	R2	9.38	69.2
	30	R3	3.67	88.0

Applicant's summary and conclusion

Validity criteria fulfilled:

no

Remarks:

The blank controls oxygen uptake rate is 18.5 mg oxygen per one gram of activated sludge (dry weight) per hour, which is below the trigger value of 20. Given the small variation between the two blank measurements (2%), the test is however deemed reliable.

Conclusions:

At all applied test concentrations, the test substance showed no significant toxic effects to the activated sludge. The respiration rates were in the same range as determined for the two blank controls.

These results should however be considered carefully as the validation criteria have not been entirely fulfilled. Given the small variation between the two blank measurements, the test is however deemed reliable.

Executive summary:

The inhibitory effect of DATIPH (4,4'-Benzylidenedianiline) on the oxygen consumption of activated sludge microorganisms of a municipal sewage treatment plant was investigated under aerobic static conditions. The inhibitory effect may include the effect on respiration and nitrification. The exposure time of the test substance on the activated sludge was 3 hours.

The nominal concentrations of DATIPH were 10, 100 and 1000 mg/l, respectively.

No chemical analysis of test concentrations was conducted. For the estimation of the effective concentration leading to 20, 50, and 80% respiration inhibition (EC₂₀, EC₅₀ and EC₈₀) the nominal content of the test substance was used, assuming the test compound to be stable in water over 3 h.

At all applied test concentrations, the test substance showed no significant toxic effects to the activated sludge. The respiration rates were in the same range as determined for the two blank controls.

The EC₂₀, EC₅₀and EC₈₀values of DATIPH were estimated to be >1000 mg/l.

These results should however be considered carefully as the validation criteria have not been entirely fulfilled. Given the small variation between the two blank measurements, the test is however deemed reliable.

All concentrations of test substance are given in total nominal content (dissolved and undissolved).