m-phenylenebis(methylamine)

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to microorganisms

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

The definitive study was performed between 1997-08-14 and 1997-108-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

other: the German Water Hazard Classification Scheme (Bewertung Wassergefaehrdender Stoffe LTWS - Nr 10) and ISO 10712 "Determination of the inhibitory effect of water constituents on bacteria (Pseudomonas cell multiplication inhibition test)"

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection 1996-01-22; Date of signature 1996-02-27

Analytical monitoring:

no

Details on sampling:

Samples of the bacterial populations were removed after approximately 16 hours and absorbance values determined for each control and treatment group.

Vehicle:

no

Details on test solutions:

For the purpose of the definitive study the test material was prepared by a direct dispersion in sterile reverse osmosis water.

An amount of test material (512 mg) was dispersed in sterile reverse osmosis water and the volume adjusted to 200 mL to give a 2560 mg/L stock solution. Serial dilutions were made from this stock solution to produce further stock solutions of 1280, 640, 320, 160, 80, 40, 20, 10, 5.0, 2.5 and 1.25 mg/L. To an aliquot (80mL) of each stock solution, nutrient stock solutions and bacterial suspension were added (see Appendix IV, below) to give the required test concentration range of 1.0, 2.0, 4.0, 8.0, 16, 32, 64, 128, 256, 512, 1024, 2048 mg/L.

Following the recommendations of the German Water Hazard Classification Scheme and ISO 10712, three identical test concentration ranges were produced as above from three separate weighings of the test material.

Analysis of the concentration, homogeneity and stability of the test material in the test solutions were not appropriate to the test guideline.

Test organisms (species):

Pseudomonas putida

Details on inoculum:

Approximately 18 hours prior to commencing the test, an aqueous suspension of Pseudomonas putida was produced by adding pre-culture medium (see appendx III, below) to a stock culture of the bacterium and gently shaking in order to wash the bacterial cells off the solid medium. The resultant suspension was dispersed into a sterile flask plugged with sterile non-absorbent cotton wool and incubated at 25 ± 1 degree Centrigade (LMS cooled incubator).

After the intial incubation period of approximately 18 hours, the bacterial suspension had a turbidity of approximately 100 Formazine Turbudity Units (FTU) (see appendix V, attached). An aliquot (50mL) of the 100 FTU bacterial suspension was added to 450mL of pre-culture medium and incubated at 25 ± 1 degrees Centigrade.

Test type:

static

Water media type:

not specified

Limit test:

no

Total exposure duration:

16 h

Post exposure observation period:

No data

Hardness:

No data

Test temperature:

25 ± 1 degrees Centigrade

pH:

No data

Dissolved oxygen:

No data

Salinity:

No data

Nominal and measured concentrations:

The test concentrations to be used in the definitive study were determined by a preliminary range-finding study. The range-finding study was conducted by exposing Pseudomonas putida to a series of nomial test concentrations of 1.0, 10, 100, 1,000 and 10,000 mg/L for a period of approximately 17 hours.

Based on the results of the range-finding study the following test concentrations were assigned to the definitive study 1.0, 2.0, 4.0, 8.0, 16, 32, 64, 128, 256, 512, 1024 and 2048 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 mL sterile glass conical flasks plugged with sterile non-absorbant cotton wool

Reference substance (positive control):

yes

Duration:

16 h

Dose descriptor:

EC10

Effect conc.:

24 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Duration:

16 h

Dose descriptor:

EC50

Effect conc.:

130 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Duration:

16 h

Dose descriptor:

NOEC

Effect conc.:

16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Details on results:

Definitive study

Mean absobance values from the exposure of Pseudomonas putida to the test material are given in Table 1 (see below) and percentage inhibition of growth is given in Table 2 (see below).

Statistical analysis of the absorbance values was carried out for the control and all test concentrations using a Students t-test. There were no statistically significant differences between the control and the 1.0, 2.0, 4.0, 8.0 and 16 mg/L test concentrations (P >= 0.05), however all other test concentrations were significantly different (P < 0.001) and thereoire the "No observed effect concentration" (NOEC) is given as 16 mg/L.

The following data show that the innoculum used in the control multiplied by a factor of 148 during the test in line with ISO guideline that states the mutliplication must be at least by a factor of 100 after 16 hours:

Mean absorbance of control at zero hours: 0.008
Mean absorbance of control at 16 hours: 1.186

Results with reference substance (positive control):

No data

Reported statistics and error estimates:

A Students t-test was carried out on the absorbance values data after approximately 16 hours exposure for the control and all test concentrations to determine any statistically significant differences between test and control groups.

Range-finding study

The mean absorbance values from the exposure of Pseudomonas putida to the test material during the range-finding study are given in Table 1:

Table 1 - Mean absorbance values from the range-finding study

Nominal Concentration (mg/L)	Absorbance Value
	0 hours	16 hours
Control	0.008	1.193
1.0	-	1.185
10	-	1.186
100	-	1.159
1000	-	0.014
10000	-	0.006
- indicates that a value was not determined

The results showed no effect on growth at the test concentrations of 1.0, 10 and 100 mg/L. However, growth was observed to be reduced at 1000 and 10000 mg/L.

Based on this information, test concentrations of 1.0, 2.0, 4.0, 8.0, 16, 32, 64, 128, 256, 512, 1024 and 2048 mg/L were selected for the main study.

Definitive study

Mean absorbance values from the exposure of Pseudomonas putida to the test material are given in Table 2 and percentage inhibition of growth is shown in Table 3:

Table 2 - Mean absorbance values from the definitive study

Nominal Concentration (mg/L)	Absorbance Value
	0 hours	16 hours
Control	0.008	1.186
1.0	-	1.193
2.0	-	1.187
4.0	-	1.177
8.0	-	1.140
16	-	1.114
32	-	0.0893
64	-	0.0855
128	-	0.654
256	-	0.241
512	-	0.105
1024	-	0.069
2048	-	0.008
- indicates that a value was not determined

Table 3 - Inhibition of growth

Nominal Concentration (mg/L)	H Values (%)
1.0	0
2.0	0
4.0	1
8.0	4
16	6
32	25
64	28
128	45
256	80
512	92
1024	95
2048	100
EC10from Figure 1 (attached) = 24 mg/L
EC50from Figure 1 (attached) = 130 mg/L

From the data given in Tables 2 and 3 (above), it was concluded that the growth of Pseudomonas putida (NCIMB 8248) was affected by the presence of the test material over the 16 hour exposure period.

Accordingly, a German Water Hazard Classification Scheme evaluation number of 4.6 was determined from the data.

Validity criteria fulfilled:

yes

Conclusions:

The effect of Meta-Xylenediamine on the growth of Psudomonas putida over a 16 hour period gave an EC10 of 24 mg/L and an EC50 of 130 mg/L. The EC10 of 24mg/L correponds to an evaluation number (Bewertungszahl, BWZ) for the German Water Hazard Classification Scheme of 4.6. Based on the results of statistical tests, the NOEC was considered to be 16 mg/L.

Reference 2

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-01-27 to 2004-01-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC Directive 88/302/EEC 18 November, 1987 (Activated Sludge Respiration Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

date of inspection: 2001-06-26; Date of signature: 2001-09-24

Analytical monitoring:

no

Details on sampling:

No data

Vehicle:

no

Details on test solutions:

See below

Test organisms (species):

other: Activated sludge from laboratory wastewater plant treating municipal sewage

Details on inoculum:

The inoculum was washed, brought to a concentration of 5 g/L dry substance and aerated during the night. 50 mL were added to a total volume of 250 mL to obtain a concentration of 1g/L dry substance in the test.

Start of study: 27/01/2004
Experimental starting date: 27/01/2004
Experimental completion date: 29/01/2004
Incubation and measurement: 29/01/2004
Incubation time: 30 min
Test temperature: 20 ± 2 degrees Centigrade
Test vessels: Erlenmeyer-vessel (nominal value 250 mL)
Test volume: 250 mL
synthetic medium: 8mL/vessel 100-fold concentrated OECD medium
Oxygen concentration during aeration: > 2.5mg/L
Oxygen concentration immediately before measurement: >6.5 mg/L
Duration of the measurement of oxygen consumption: 8-10 min

Test type:

static

Limit test:

no

Total exposure duration:

30 min

Post exposure observation period:

No data

Hardness:

No data

Test temperature:

20 ± 2 degrees Centigrade

pH:

Please refer to Table 2 (below)

Dissolved oxygen:

Oxygen concentration during aeration: > 2.5 mg/L
Oxygen concentration immediately before measurement: > 6.5 mg/L
Duration of measurement of oxygen consumption: 8-10 min

Salinity:

No data

Nominal and measured concentrations:

Please refer to details presented in the section entitled 'any other information on materials and methods incl. tables' (below)

Details on test conditions:

Incubation time: 30 min
Test vessels: Erlenmeyer-vessel (nominal volume 250 mL)
Test volume: 250 mL
Synthetic medium: 8 mL/vessel 100 -fold concentrated OECD medium

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

30 min

Dose descriptor:

other: EC20

Effect conc.:

ca. 500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: respiration inhibition

Duration:

30 min

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: respiration inhibition

Duration:

30 min

Dose descriptor:

other: EC80

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: respiration inhibition

Details on results:

The EC-values are based on added nominal concentrations and not on analytically determined values. The nominal concentrations which indicate a respiration inhibition of 20, 50, 80% can be taken from the graph of the inhibition curve (please see the attached background material).

Validity criteria
- Deviation of blank controls < 15%: yes
- EC50 of 3,5-dichlorophenol in the range 5-30 mg/L: yes
- Test is valid: yes

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: After 30 minutes of incubation:

[EC20 = ca. 2.5 mg/L], [EC50 = ca. 16 mg/L], [EC80 > 100mg/L*]

* highest concentration tested was 100mg/L

Reported statistics and error estimates:

No data

Table 1 - Measured data: blank control (BC) and test substance (TS) after 30 minutes incubation time

mv BC = mean value of blank control

Vessel no.:	BC1	BC2	BC3	Mv BC	TS1	TS2	TS3
Concentration of test substance (mg/L)	-	-	-	-	1000	496	248
Added volume of stock solution (mL/vessel)	-	-	-	-	125.0	62.0	31.0
Direct addition (mg/vessel)	-	-	-	-	-	-	-
Synthetic medium (mL/vessel)	8	8	8	-	8	8	8
Inoculum (mL/vessel)	50	50	50	-	50	50	50
Filled up to the total volume with deionized water (mL/vessel)	250	250	250	-	250	250	250
Oxygen concentration start (mg O2/L)	7.5	7.6	7.8	-	7.4	7.4	7.8
Oxygen concentration after 6 min (mg O2/L)	4.8	5.0	5.1	-	5.8	5.2	5.5
Oxygen concentration after 6 min (mg O2/L*6min)	2.7	2.6	2.7	2.7	1.6	2.2	2.3
Oxygen consumption rate (mg O2/L*h)	27	26	27	27	16	22	23
Specific oxygen consumption rate (mg O2/g*h)	27	26	27	27	16	22	23
Change in oxygen consumption rate compared with the blank control (%)	-	-	-	-	41	19	15

Table 2 - pH-values: blank control (BC) and test substance (TS)

Vessel no.:	BC1	BC2	BC3		TS1	TS2	TS3
pH-value before adding the inoculum before correction:	7.3	7.3	6.8		6.7	6.8	6.8
After correction	7.3	7.3	7.2		7.1	7.2	7.3
pH-value after an incubation time of 30 min	-	-	-		7.8	-	-

Table 3 - Measured data: reference susbtance (RS) after 30 minutes incubation time

Test vessel number	mv BC	RS1	RS2	RS3
Concentration of substance (mg/L)	-	1	10	100
Added volume of stock solution (mL/vessel)	-	0.5	5.0	50.0
Synthetic medium (mL/vessel)	-	8	8	8
Inoculum (mL/vessel)	-	50	50	50
Filled up to the total volume with deionized water (mL/vessel)	-	250	250	250
Oxygen concentration start (mg O2/L)	-	7.6	8.0	8.6
Oxygen concentration after 6 min (mg O2/L)	-	5.0	6.5	8.0
Oxygen consumption rate (mg O2/L*6 min)	2.7	2.6	1.5	0.6
Oxygen consumption rate (mg O2/L*h)	27	26	15	6
Specific oxygen consumption rate (mg O2/g*h)	27	26	15	6
Change in oxygen consumption rate compared to the blank control (%)	-	4	44	78

Table 4 - pH values: reference substance (RS)

 

Vessel no.:	RS1	RS2	RS3
pH-value before adding the inoculum before correction:	7.3	7.1	7.1
after correction:	7.3	7.1	7.1

Table 5 - Effective concentrations: EC values reference substance (RS) after 30 minutes incubation time

	EC20 mg/L	EC50 mg/L	EC80 mg/L	Highest concentration tested mg/L
Reference substance	About 2.5	About 16	> 100	100

Validity criteria fulfilled:

yes

Conclusions:

Test results were reported as EC20 (30 min) about 500 mg/L (nominal), EC50 (30 min) > 1000 mg/L (nominal) and EC80 (30 min) > 1000 mg/L (nominal).

The EC20 in the activated sludge respiration inhibition test is > 1000 mg/L. Disturbances in the biodegradation process of activated sludge are not to be expected if the test substance is correctly introduced into adapted wastewater treatment plants at low concentrations.

Description of key information

EC50 (30 min) > 1000 mg/L on (OECD 209)

EC50 (16 h) 130 mg/L (ISO 10712)

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

Additional information

Two GLP studies are available to describe the effect of the test substance on microorganisms in activated sludge from wastewater treatment plants. The report prepared by Bachner (2004) has been flagged as the key study despite the approximate LC50 value because OECD 209 permits respiration rate to be measured after 30 minutes, or 3 hours, or both. In addition, ECHA guidance on information requirements and chemical safety assessment Chapter R.10: Characterisation of dose [concentration]-response for environment prefers OECD 209 over ISO 10712 when calculating the PNEC for microorganisms in sewage treatment plants.