Reaction mass of N1,N1'-(methanediyldibenzene-4,1 -diyl)diaryldiamine and 2-{4-[(4-aminophenyl)amino]benzyl}-N4-(4-{4-[(4-aminophenyl)amino]benzyl}phenyl)aryldiamine

Toxicity to terrestrial plants

Link to relevant study record(s)

Reference

Endpoint:

toxicity to terrestrial plants: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 December 2014 to 05 August 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: HJ/T 153-2004, The guidelines for the testing of chemicals [S]. Beijing: SEPA, 2004

Deviations:

yes

Remarks:

mung bean and carrot were replaced with soybean and radish with no impact on the outcome or integrity of the study (see below)

Qualifier:

according to guideline

Guideline:

other: CRC-MEP. The Guidelines for the Testing of Chemicals, Degradation and AccumulationfM]. 2nd edition. Beijing: China Environment Press. 2013: 459-463.

Deviations:

yes

Remarks:

mung bean and carrot were replaced with soybean and radish with no impact on the outcome or integrity of the study (see below)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

SAMPLING
- The initial concentration of test substance at the start of the test was determined by UPLC-PDA.
- Samples at different concentrations were prepared by adding the same volume of stock solution as used in preparation of test substrates to another 20 g quartz sand. After evaporation, the solvent at room temperature, 100 mL acetonitrile was added and then ultrasonic extraction was conducted.
- Samples of blank control, solvent control and 10 mg/L: 50.0 mL acetonitrile extract was filtrated through 0.22 µm membrane (discarding the initial 10.0 mL filtrates) and analysed by UPLC-PDA.
- Samples of 100 mg/L: 5.00 mL acetonitrile extract were diluted to 50.0 mL (diluted 10 fold) then filtrated through 0.22 µm membrane (discarding the initial 10.0 mL filtrates) and analysed by UPLC-PDA.
- Samples of 1000 mg/L: 0.50 mL acetonitrile extract were diluted to 50.0 mL (diluted 100 fold) then filtrated through 0.22 µm membrane (discarding the initial 10,0 mL filtrates) and analysed by UPLC-PDA.
- The initial concentration of test item was calculated by total amount of test substance in the 100 mL acetonitrile extract divided by 50.0 mL (volume of the deionised water).

Vehicle:

yes

Remarks:

acetonitrile

Details on preparation and application of test substrate:

PREPARATION OF TEST SUBSTRATES
- Stock solution (10000 mg/L) was prepared by directly dissolving 1.0001 g test substance into 100 mL dimethylformamide.
- The test medium was prepared by adding appropriate test solution to 10 g quartz sand.
- The solvent was evaporated to dry at room temperature then 190 g quartz sand was thoroughly incorporated and 50 mL deionized water was added.
- Details of media preparation are given in the table below.

Species:

Brassica campestris var. chinensis

Details on test organisms:

Batch S20140807BC

Species:

Lactuca sativa

Details on test organisms:

Batch S20140807LS

Species:

other: Oryza sativa L

Details on test organisms:

Batch S20140807O

Species:

other: Brassica oleracea

Details on test organisms:

Batch S20140807BO

Species:

Cucumis sativus

Details on test organisms:

Batch S20140807C

Species:

Zea mays

Details on test organisms:

Batch S20140807Z

Species:

other: Sesamum indicum

Details on test organisms:

Batch S20140807S

Species:

Lycopersicon esculentum

Details on test organisms:

Batch S20140807LE

Species:

other: Glycine max

Details on test organisms:

Batch S20140807G

Species:

Raphanus sativus

Details on test organisms:

Batch S20140807R

Test type:

seed germination/root elongation toxicity test

Study type:

laboratory study

Substrate type:

other: quartz sand

Limit test:

no

Test temperature:

25 ± 1 °C

pH:

7.56 TO 7.71

Moisture:

80 ± 5 % humidity

Details on test conditions:

SELECTION OF SPECIES
- Ten species were chosen as the test organism because of their economic or ecological importance in China.
- The seeds were hold in refrigeration facilities in cold storage (5 °C) in moisture-proof containers at seed moisture contents of less than 10 percent.
- The seed was brought in from Nanjing Research Institute of Vegetables and Flowers. The information on seed lot, the seed year or growing season collected and germination percentage was provided by the supplier of the seed.
- Only untreated seed (not treated with fungicides, repellents, etc.) taken from the same lot and year or season of collection was used in the test.
- Seeds were separated into appropriate size classes, and that size class containing the most seed was used exclusively for the test.
- Damaged seeds were discarded.

APPARATUS
- Digital multi-parameter meter (HQ30d, HACK Corporation)
- Climatic chambers (RXZ-600B, Ningbo Jiangnan Instrument Factory)
- Refrigeration facilities (BCD-218, Germany’s Siemens)
- Petri dishes (Diameter = 200 mm, Dahua glassware ltd., Xuzhou)
- Vernier calliper (16EX, Mahr, Germany)
- Electronic balance (JM-A20002, Zhejiang Yuyao Jinging Weighing Scale Co., Ltd.; MS105DU, METTLER TOLEDO)
- ACQUTTY Ultra Performance LC (Waters USA)

TEST CONCENTRATIONS
- Nominal concentrations of 10, 100 and 1000 mg/L test substance were used in range-finding test.
- A blank control and a solvent control without the test substance were run at the same time.
- There were no significant phytotoxicity observed in the range-finding test for Brassica chinensis L. (Cabbage), Lactuca sativa (lettuce), Brassica oleracea (Kohlrabi), Cucumis sativus (cucumber), Zea mays (corn), Sesamum indicum (sesame), Lycopersicon esculentum (tomato) (p > 0.05).
- Although there were significant phytotoxicity observed in the range-finding test for Oryza sativa L. (Rice), Glycine max (soybean) and Raphanus sativus (Radish) (p < 0.05), the inhibition rates for root elongation were < 50%. Therefore, no definitive testing was carried out.

TEST PERFORMANCE
- Fresh test solutions were added to glass dishes that have been filled with 200 g clean quartz sand.
- The seed was then positioned on the substrate allowing adequate room for anticipated growth. The radicle end of the seed was aligned in the direction of this growth.
- dishes were sealed with preservative film with some holes.
- The dishes were then placed in a climatic chamber.
- During the test, the following conditions were maintained: complete darkness; temperature: 25 ± 1 °C; humidity 80 ± 5 %.
- Seeding densities were 15 seeds /dish for range-finding test (no replicates).
- The test period ended when at least 65 % of the control seed germinated and developed roots at least 20 mm long. When both conditions were satisfied, the mean number of seeds germinating and mean root length per treatment (and control) were determined.
- EC50s for germination and root elongation were determined for each species tested and reported along with their 95 % confidence limits.

OBSERVATIONS AND MEASUREMENTS
- Since seed germination and root elongation varied, the exposure duration for each test species is shown in the table below.
- When the exposure ended, the number of seeds that had germinate was counted and root lengths were measured.
- All root elongation measurements for a given species were made sequentially before proceeding to the next species.
- Root length was measured from the transition point between the hypocotyl and root to the tip of the root.
- Temperature in the germination facility was reported at the start and the end of the test and the pH of the test solutions was recorded at the initiation of the test.

VALIDITY OF THE TEST
- Control group: One dilution-medium control (water) and one solvent control series were also run in addition to
the treatment series.
- Validity of test result: During the whole test period, more than 65 percent of the control seed germinated at the end of test with the root length greater than 20 mm; therefore the test was considered valid.

Nominal and measured concentrations:

10, 100 and 1000 mg/L

Reference substance (positive control):

no

Key result

Species:

Brassica campestris var. chinensis

Duration:

5 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

phytotoxicity

Remarks on result:

other: germination and root elongation

Key result

Species:

Lactuca sativa

Duration:

5 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

phytotoxicity

Remarks on result:

other: germination and root elongation

Key result

Species:

Oryza sativa

Duration:

5 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

phytotoxicity

Remarks on result:

other: germination and root elongation

Key result

Species:

other: Brassica oleracea

Duration:

5 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

phytotoxicity

Remarks on result:

other: germination and root elongation

Key result

Species:

Cucumis sativus

Duration:

5 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

phytotoxicity

Remarks on result:

other: germination and root elongation

Key result

Species:

Zea mays

Duration:

5 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

phytotoxicity

Remarks on result:

other: germination and root elongation

Key result

Species:

other: Sesamum indicum

Duration:

6 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

phytotoxicity

Remarks on result:

other: germination and root elongation

Key result

Species:

Lycopersicon esculentum

Duration:

6 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

phytotoxicity

Remarks on result:

other: germination and root elongation

Key result

Species:

other: Glycine max

Duration:

5 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

phytotoxicity

Remarks on result:

other: germination and root elongation

Key result

Species:

Raphanus sativus

Duration:

5 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 other: mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

phytotoxicity

Remarks on result:

other: germination and root elongation

Details on results:

TEST CONDITIONS
- The pH and temperature of the control and treatment groups during the test are shown in Tables 1 and 2 respectively (attached).
- During the whole test period, the pH values of the control and test media freshly prepared were between 7.56 and 7.71, and the temperature in the germination facility was maintained in the range of 24 to 25 °C.

CALIBRATION CURVE
- A series of standard solutions with concentration at 0.50, 1.00, 2.00, 5.00 and 10.0 mg/L were measured under the UPLC-PDA conditions described above.
- Based on the test result, a linear regression equation was obtained between the concentration and the area of the peak: A = 139059c-55817, with good linearity of r2 = 0.9988, where > 4 represents peak area (µV*s); and c
is the concentration of test substance (mg/L) (see Figure 1, attached).
- The results show that linearity for concentration range of 0.50 mg/L to 10.0 mg/L is good.

DETECTION LIMIT AND QUANTITATION LIMIT
- The detection limit was 0.06mg/L (S/N > 3), and the quantitation limit was 0.20 mg/L (S/N > 10).

RESULTS OF TEST CONCENTRATION ANALYSIS
- The analysed results of the test concentration were listed in Table 1 (attached).
- According to the test result, the analysed concentration of 10 mg/L, 100 mg/L and 1000 mg/L were 9.31 mg/L, 95.5 mg/L and 903 mg/L respectively.

EFFECT ON GERMINATION RATES AND ROOT ELONGATION
- Table 3 (attached) shows germination rates and germination inhibition rates observed for the seeds of different species.
- Tables 4 to 13 (attached) show root lengths and root lengths inhibition rates observed for the seeds of different species.
- At the end of the test, over 86.7 percent of the control seeds germinated and the roots developed were longer than 20 mm. Compared with the germination rates of the solvent control groups, the germination rates of the highest concentration group (1000 mgL) were < 50%.
- The mean root length for each concentration was compared with negative and solvent controls using the multiple comparison method of Dunnet's tests (ANOVA, SPSS 17.0). The results showed that no significant difference was observed for Brassica chinensis L. (Cabbage), Lactuca sativa (lettuce), Brassica oleracea (Kohlrabi), Cucumis sativus (cucumber) , Zea mays (corn), Sesamum indicum (sesame), Lycopersicon esculentum (tomato) (p > 0.05). - Although there were significant phytotoxicity observed in the range-finding test for Oryza sativa L. (Rice), Glycine max (soybean) and Raphanus sativus (Radish) (p < 0.05), the inhibition rates for root elongation
were < 50%.
- The germination rates for each concentration were compared with the solvent control by analysis of Chi-square Test (SPSS 17.0). The results showed that the effect of 10 mg/L, 100 mg/L and 1000 mg/L on germination rates of the 10 plant seeds showed no significant difference (p > 0.05).

Reported statistics and error estimates:

DATA PROCESSING
- For the estimation of the lowest observed effect concentration (LOEC) at P < 0.05, and hence the no observed effect concentration (NOEC), for effects of the chemical on root elongation, the mean root length for each concentration were compared with the control mean using a multiple comparison method of Dunnett’s tests (ANOVA, SPSS 17.0).
- For the estimation of the lowest observed effect concentration (LOEC) at P < 0.05, and hence the no observed effect concentration (NOEC), for effects of the chemical on germination, Chi-square Test was used (SPSS 17.0).
- Trimmed Spearman-Karber Method (Version 1.5, USEPA) was used to calculate the EC50 and EC50 confidence limits. This method was not carried out because of the low toxicity of the test substance.

Validity criteria fulfilled:

yes

Conclusions:

The LOEC values for germination were determined to be > 1000 mg/L in all ten species, NOEC values for germination were 1000 mg/L in all ten species and EC50 values for germination were > 1000 mg/L in all ten species. With respect to root elongation, LOEC values were determined to be > 1000 mg/L for all species apart from rice, soybean and radish where the LOEC was reported as 1000 mg/L. The NOEC for root elongation was 1000 mg/L in all species apart from rice, soybean and radish where the NOEC was reported as 100 mg/L. The EC50 value for root elongation was > 1000 mg/L in all ten species. No definitive testing was performed because the inhibition rates for root elongation were < 50 %.

Executive summary:

GUIDELINE

Under valid conditions, the phytotoxicity of the test substance to 10 terrestrial plant species was conducted according to “The guidelines for the testing of chemicals” (HJ/T 153-2004).

 

METHODS

Ten plant species including Brassica chinensis L. (Cabbage), Lactuca sativa (Lettuce), Oryza sativa L. (Rice), Brassica oleracea (Kohlrabi), Cucumis sativus (Cucumber), Zea mays(Corn),Sesamum indicum (Sesame), Lycopersicon esculentum (Tomato), Glycine max (Soybean),Raphanus sativus (Radish) (15 seeds/group/species) were exposed to 10, 100 and 1000 mg/L of the test substance. A blank control and a solvent control without the test substance were run at the same time. During the whole test period, the pH values of the freshly prepared control and Test Media were between 7.56 and 7.71, and the temperature in the germination facility was maintained in the range of 24 to 25 °C. At the end of the test, more than 65 % of the seeds in blank control and solvent control germinated, and the roots developed were longer than 20 mm. The study met the acceptability criteria prescribed by the protocol and the test was considered valid.

 

RESULTS
The initial concentration of test item in the test solution at the start of the test was determined by UPLC-PDA. According to the test result, the analysed concentration of 10 mg/L, 100 mg/L and 1000 mg/L were 9.31 mg/L, 95.5 mg/L and 903 mg/L respectively. There was no significant phytotoxicity observed in the range-finding test for Brassica chinensis L. (Cabbage), Lactuca sativa (lettuce), Brassica oleracea (Kohlrabi), Cucumis sativus (cucumber), Zea mays (corn), Sesamum indicum (sesame), Lycopersicon esculentum (tomato) (p > 0.05). Although there were significant phytotoxicity observed in the range-finding test for Oryza sativa L. (Rice), Glycine max (soybean) and Raphanus sativus (Radish) (p < 0.05), the inhibition rates for root elongation were < 50%. Therefore, no definitive testing was carried out. The results showed that under valid test conditions, the LOEC, NOEC and EC50 for all ten plant species tested were as shown in the table below.

 

Seed	Germination LOEC (mg/L)	Germination NOEC (mg/L)	Germination EC50 (mg/L)	Root elongation LOEC (mg/L)	Root elongation NOEC (mg/L)	Root elongation EC50 (mg/L)
Cabbage	> 1000	1000	> 1000	> 1000	1000	> 1000
Lettuce	> 1000	1000	> 1000	> 1000	1000	> 1000
Rice	> 1000	1000	> 1000	1000	100	> 1000
Kohlrabi	> 1000	1000	> 1000	> 1000	1000	> 1000
Cucumber	> 1000	1000	> 1000	> 1000	1000	> 1000
Corn	> 1000	1000	> 1000	> 1000	1000	> 1000
Sesame	> 1000	1000	> 1000	> 1000	1000	> 1000
Tomato	> 1000	1000	> 1000	> 1000	1000	> 1000
Soybean	> 1000	1000	> 1000	1000	100	> 1000
Radish	> 1000	1000	> 1000	1000	100	> 1000

The measured concentration for a nominal concentration of 1000 mg/L was 903 mg/L

The measured concentration for a nominal concentration of 100 mg/L was 95.5 mg/L

CONCLUSION

The LOEC values for germination were determined to be > 1000 mg/L in all ten species, NOEC values for germination were 1000 mg/L in all ten species and EC50 values for germination were > 1000 mg/L in all ten species. With respect to root elongation, LOEC values were determined to be > 1000 mg/L for all species apart from rice, soybean and radish where the LOEC was reported as 1000 mg/L. The NOEC for root elongation was 1000 mg/L in all species apart from rice, soybean and radish where the NOEC was reported as 100 mg/L. The EC50 value for root elongation was > 1000 mg/L in all ten species. No definitive testing was performed because the inhibition rates for root elongation were < 50 %.

Description of key information

Under valid conditions, the phytotoxicity of the test substance to ten terrestrial plant species was conducted according to “The guidelines for the testing of chemicals” (HJ/T 153-2004). The LOEC values for germination were determined to be > 1000 mg/L in all ten species, NOEC values for germination were 1000 mg/L in all ten species and EC50 values for germination were > 1000 mg/L in all ten species. With respect to root elongation, LOEC values were determined to be > 1000 mg/L for all species apart from rice, soybean and radish where the LOEC was reported as 1000 mg/L. The NOEC for root elongation was 1000 mg/L in all species apart from rice, soybean and radish where the NOEC was reported as 100 mg/L. The EC50 value for root elongation was > 1000 mg/L in all ten species. No definitive testing was performed because the inhibition rates for root elongation were < 50 %.

Additional information

GUIDELINE

Under valid conditions, the phytotoxicity of the test substance to ten terrestrial plant species was conducted according to “The guidelines for the testing of chemicals” (HJ/T 153-2004).

 

METHODS

Ten plant species including Brassica chinensis L. (Cabbage), Lactuca sativa (Lettuce), Oryza sativa L. (Rice), Brassica oleracea (Kohlrabi), Cucumis sativus (Cucumber), Zea mays (Com), Sesamum indicum (Sesame), Lycopersicon esculentum (Tomato), Glycine max (Soybean), Raphanus sativus (Radish) (15 seeds/group/species) were exposed to 10, 100 and 1000 mg/L of the test substance. A blank control and a solvent control without the test substance were run at the same time. During the whole test period, the pH values of the freshly prepared control and Test Media were between 7.56 and 7.71, and the temperature in the germination facility was maintained in the range of 24 to 25 °C. At the end of the test, more than 65 % of the seeds in blank control and solvent control germinated, and the roots developed were longer than 20 mm. The study met the acceptability criteria prescribed by the protocol and the test was considered valid.

 

RESULTS
The initial concentration of test item in the test solution at the start of the test was determined by UPLC-PDA. According to the test result, the analysed concentration of 10 mg/L, 100 mg/L and 1000 mg/L were 9.31 mg/L, 95.5 mg/L and 903 mg/L respectively. There was no significant phytotoxicity observed in the range-finding test for Brassica chinensis L. (Cabbage), Lactuca sativa (lettuce), Brassica oleracea (Kohlrabi), Cucumis sativus (cucumber), Zea mays (com),Sesamum indicum (sesame), Lycopersicon esculentum (tomato) (p > 0.05). Although there were significant phytotoxicity observed in the range-finding test for Oryza sativa L. (Rice), Glycine max (soybean) and Raphanus sativus (Radish) (p < 0.05), the inhibition rates for root elongation were < 50%. Therefore, no definitive testing was carried out. The results showed that under valid test conditions, the LOEC, NOEC and EC50 for all ten plant species tested were as shown in the table below.

 

Seed	Germination LOEC (mg/L)	Germination NOEC (mg/L)	Germination EC50 (mg/L)	Root elongation LOEC (mg/L)	Root elongation NOEC (mg/L)	Root elongation EC50 (mg/L)
Cabbage	> 1000	1000	> 1000	> 1000	1000	> 1000
Lettuce	> 1000	1000	> 1000	> 1000	1000	> 1000
Rice	> 1000	1000	> 1000	1000	100	> 1000
Kohlrabi	> 1000	1000	> 1000	> 1000	1000	> 1000
Cucumber	> 1000	1000	> 1000	> 1000	1000	> 1000
Corn	> 1000	1000	> 1000	> 1000	1000	> 1000
Sesame	> 1000	1000	> 1000	> 1000	1000	> 1000
Tomato	> 1000	1000	> 1000	> 1000	1000	> 1000
Soybean	> 1000	1000	> 1000	1000	100	> 1000
Radish	> 1000	1000	> 1000	1000	100	> 1000

The measured concentration for a nominal concentration of 1000 mg/L was 903 mg/L

The measured concentration for a nominal concentration of 100 mg/L was 95.5 mg/L

CONCLUSION

The LOEC values for germination were determined to be > 1000 mg/L in all ten species, NOEC values for germination were 1000 mg/L in all ten species and EC50 values for germination were > 1000 mg/L in all ten species. With respect to root elongation, LOEC values were determined to be > 1000 mg/L for all species apart from rice, soybean and radish where the LOEC was reported as 1000 mg/L. The NOEC for root elongation was 1000 mg/L in all species apart from rice, soybean and radish where the NOEC was reported as 100 mg/L. The EC50 value for root elongation was > 1000 mg/L in all ten species. No definitive testing was performed because the inhibition rates for root elongation were < 50 %.