Benzenamine, N-phenyl-, reaction products with 2,4,4-trimethylpentene

Toxicity to soil microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil microorganisms

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Read across from a structurally very similar substance.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Deviations:

yes

Remarks:

The test system (soil) was stored in the cooling chamber at 5.8 °C to 9.5 °C instead of the required range of 4 ± 2 °C, due to technical problems. An influence on the test result due to this deviation can be excluded.

Qualifier:

according to guideline

Guideline:

EU Method C.21 (Soil Microorganisms: Nitrogen Transformation Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

not required

Vehicle:

yes

Remarks:

Acetone

Details on preparation and application of test substrate:

Application of the Test substance:
The required aliquots of the stock solution with acetone were applied to the scheduled amount of quartz sand. To equalise the volume of acetone in each test substance -quartz sand mixture to the highest volume used in the solvent control and in 1000 mg/kg Dw test assay, extra acetone was supplied whenever needed.Thereafter, the organic solvent was removed by evaporation. The test substance / quartz sand mixtures were added to approximately 1000 g soil (dry weight) afterwards.
REASON FOR THE CHOICE OF THE APPLICATION:
Based on the specified water solubility the application of the test substance via an aqueous stock solution is not possible. The water solubility is not high enough to prepare a sufficient stock solution with deionized water. In a pre-test, the test substance was miscible with acetone and therefore, acetone was chosen as the solvent to dissolve the test substance. Test substance dissolved in acetone, added through a carrier was selected as the application method. The carrier, loaded with the test substance aliquots can be distributed evenly in the test system.

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

19.9 °C – 20.8 °C

Moisture:

40 ±5% of the WHCmax

Organic carbon content (% dry weight):

0.68

Nitrogen content (% dry weight):

0.08

Details on test conditions:

TEST SYSTEM-
Soil type: 2.3- Batch no.: F 2.3 0618-
Treatment: The soil was not treated with crop protection products in the sampling year and during the four former years. No fertiliser application was made in the last three years from the date of soil sampling. The information regarding the soil was provided by the supplier of the soil. The analyses were performed according to GLP. The following parameters were measured in the test facility according to GLP without GLP status.-
Sampling depth: Approximately 0 to 20cm
Sampling quantity: Approximately 1200kg
Sampling date: 07 Feb 2018-
Weather condition on sampling: Cloudy with air temperature of 4°C-
Preparation of the soil by the supplier: Soil prepared at 22 °C as following: drying for six days at room temperature until the soil is sievable; Pre-sieving with 10 mm screen and last sieving with 2 mm screen on 7th day; water content: 4.82g/100g dry weight; dry matter: 95.40%.-
Supplier: Landwirtschaftliche Untersuchungs- und Forschungsanstalt Speyer, Obere Langgasse 40, 67346 Speyer, Germany.
Arrival in the testing facility: 19 Feb 2018-
Storage: After arrival in the laboratory, the soil was stored in a cold storage room in a closed plastic bag at a temperature from 5.8 to 9.5 °C. This was outside the guideline recommended range of 4 ± 2 °C. –
Soil type according German DIN: Silty sand (uS),
pH-value (0.01M CaCl2): 5.84 ±0.6-
Cation exchange capacity: 7.5 ±0.8 meq/100g-
Water holding capacity: 35.4 ±1.0 g/100g-
Grain size: 2 mm-
Water content of the soil (measured): 4.8 g/100g Dw (mean of 2 values); dry matter: 95.40%.-
Initial microbiological biomass (measured): 398.7 ±2.3 mg carbon/kg Dw (mean of 2 values, correspond to 5.9% of carbon content of the soil)-
Pre-arrangement: Two days before start of exposure, 9432.1 g of the soil were mixed with 846 mL deionized water (the resulting water content corresponds to 40% of the water holding capacity). Afterwards, this was stored in a laboratory room at room temperature until usage. At the day of usage, the water content of the soil was again adjusted with deionized water. Then 40.1g powdered lucerne grass-green meal was added to 9137.0g of this wet soil at the start of exposure (day 0).
NITROGEN SOURCE
Luzerne green gras meal-
Supplier: Tock GmbH Futtermühle; 66798 Wallerfangen-Ihn, Weinbachstr. 18-20
- Date of delivery: 18 June 2003-
Crude protein: 17 %-
Carotene: 80 mg/kg-
Content of carbon: 41.9 g/100g-
Content of nitrogen: 2.4 g/100g-
C/N ratio: 17/1-
Size: Dried and pulverized
SAMPLING PROCEDURE
Three samples from each test assay were taken at the start of exposure (day 0), and after 7, 14 and 28 days for determination the nitrate content. For each sample, about 22.8g of soil was weighed into 250ml glass bottles and 100mL of deionized water was added into the bottle for the extraction of nitrate. The soil suspensions were shaken for about 1 hour at 150 rpm on a laboratory shaker. The temperature during the extraction were 20.0°C on day 0, 20.1°C on day 7, 20.3 °C on day 14 and 20.4°C on day 28. Aliquots of the supernatant of the suspensions were taken and centrifuged for about 30 minutes at 4500 rpm.
MEASURING METHOD
The content of nitrate was measured as nitrate-nitrogen(NO3-N) in each supernatant of the test mixtures using the analytical test kit no. LCK 339 from company Hach Lange GmbH, Germany. An appropriate amount of the supernatant was pipetted into the cuvette and mixed thoroughly by slowly inverting the cuvette few times. After 15 minutes of reaction time, the cuvettes containing the reaction mixture were placed in the photometer DR 3900 at 345 nm from company Hach Lange GmbH, Germany. According to the parameters for the test kit stored in the photometer, the content of NO3-N was evaluated.
EXPERIMENTAL PROCEDURE-
Test vessels for incubation: As test vessel for the incubation of the prepared soil, 2 L glass containers with a wide opening were used. They were covered with a perforated aluminum lid..-
Preparation of the test assays:
Quartz sand mixed with the required aliquots of test substance stock solution in acetone for each scheduled test concentration were mixed with the soil. The blending of the test mixtures was done with a mixer. No analyses for the concentration and stability of the test substance in the test medium were done as part of this study since this information is not relevant for this study type and the guidelines do not foresee analytical monitoring of the test substance in the soil. The first portion of the prepared wet soil with lucerne meal but without test substance was used as control (BC). Five portions of the same soil were mixed with the test substance - quartz sand mixture for preparing the test concentration series TS1-TS5 . For the test assay TS5-N, prepared wet soil without lucerne meal was mixed with the test substance (1000 mg/Kg Dw) – quartz sand mixture.

Nominal and measured concentrations:

nominal: 62.5 mg/kg, 125 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg soil dry weight

Reference substance (positive control):

no

Key result

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Details on results:

In a 28-day toxicity study on the soil nitrogen transformation activity, natural soil was exposed to the test substance, at nominal concentrations of 0 (blank control with solvent), 62.5 mg/kg, 125 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg (dry weight) under conditions in accordance with the guidelines. Lucerne meal was used as the additional nitrogen source. Nitrate formed during microbial processes was determined at the start of exposure (Day 0), after 7, 14 and at the end of exposure (Day 28) and were compared to control assays of the corresponding days. For the evaluation of results, decrease of nitrate in the treated replicates is expressed as an inhibition of test substance on the N mineralization process. Likewise, an increase in the nitrate in the treated samples is expressed as a stimulation effect of the test substance on the process of nitrogen oxidation in soil.

The calculated nitrate values in mg/kg Dw of the test assays with test substance were compared with the mean values of nitrate of the blank control (see below). The application of test substance caused concentration-dependent effects, namely concentration dependent increased nitrate values at day 28 and increased nitrogen transformation rates (difference of nitrate content between day 28 and day 0). A dose - response relationship (i.e., hormesis) attributed to this stimulation pattern was also observed. Only in the lower concentration test on day 28, nitrate concentration >25% compared to the control treatment was observed. In all other test concentrations, differences to the control were small and not significant.The test batches remained visually unchanged during the exposure period. The coefficient of variation in replicates of the blank solvent control at the end of the exposure was 1.9%.

There is an overall stimulation of organic nitrogen breakdown in the 62.5mg /kg dry soil weight, which is reflected in the results by increased levels of the end product of mineralization and nitrification, nitrate, by Day 28. The results in indicate a clear hormesis effect and therefore, a model assuming monotone decreasing doseresponse is not valid.
A prerequisite for calculating ECx values is an inhibitory effect. However, at the highest test concentration of 1000 mg/kg soil (dw), no negative impairment on nitrogen transformation was observed. In contrast, the application of test substance caused concentration-dependent stimulation effects, namely concentration dependent increased nitrate values at day 28 and increased nitrogen transformation rates (difference of nitrate content between day 28 and day 0). Therefore, the EC10, EC25 and EC50 values were estimated to be likely >1000 mg/kg Dw while no inhibition was found compared to the controls.
Because the degree of inhibition at 1000 mg/kg Dw was less than 25 % at the end of exposure according to the OECD guideline 216, the test item can be evaluated as having no long-term influence on nitrogen transformation in soils. On Day 28, inside the tested concentration ranges (62.5 – 1000 mg/kg), the highest stimulation (=highest nitrate evolution) up to 28.0% was noted in the lowest test concentration of 62.5 mg/kg Dw while in the 1000 mg/kg Dw test assay, a lowest stimulation of 7.5% was noted, compared to the control. At 62.5 mg/kg Dw and only on Day 28, a difference >25% in nitrate
content compared to the control could be reported.
The nitrate content determined in the test assay without Luzerne meal (TS5-N) on day 28 show a significant increase compared to the same test assay of day 0.
The pH and temperature at which the exposure was performed were within the acceptable guideline specifications. There were no morphological observations made during the incubation and the soil remained visually unchanged during exposure period.
The results in this study are consistent with all relevant validity criteria and the test is valid according to the guidelines of this study.

Reported statistics and error estimates:

The statistical evaluation of the data was carried out using ToxRat. If the % inhibition was lower than 0% it was set to 0 %. If the %inhibition was greater than 100 % it was set to 100 %. A curve was fitted between the concentration and the %inhibition via the probit model. This curve was used for the estimation of the EC10, EC25 and EC50. Appropriate weighting for the model fitting via linear transformation were used [2]. The confidence intervals were determined via normal approximation. The additional test assay TS5-N was not included in the statistical analysis. The substance has N which is in the diphenylamine-core of the structure and there is a high probability that this bond will not be broken during microbial mechanisms, at least in 28 days (EU RAR 2008 CAS 122-39-4). According to the information from sponsor, the substance is not readily biodegradable and is unlikely to release N from the central core. Therefore, it is assumed that the increasing nitrate content in the additional test assays (TS5-N) do not arise from the test substance molecule, but possibly from other sources. For these reasons the origin of the nitrate should be designated as nonspecific and cannot assigned clearly to the test substance in this test and as such, not considered for statistical evaluation.

Results of the nitrate determination at the start of exposure (day 0)

a) water content of 14.2 g/100g Dw;

b) Inhibition is indicated in negative values and stimulation is in positive values;

c) test assay without lucerne meal.

Test concentration [mg/kg Dw] nominal	Replicate	Test mixture for analyses	Eluate	Eluate	Test assay	b Change
			nitrate- nitrogen	calculated nitrate	calculated nitrate
		[g] a	[mg/L]	[mg/L]	[mg/kg Dw]	[%]
Blank control	1	22.8	5.27	23.3	117	-
Blank control	2	22.8	5.06	22.4	112	-
Blank control	3	22.8	5.42	24.0	120	-
62.5	1	22.8	5.76	25.5	128	10.0
62.5	2	22.8	5.66	25.1	126	8.3
62.5	3	22.8	5.79	25.6	128	10.0
125	1	22.8	5.76	25.5	128	10.0
125	2	22.8	5.46	24.2	121	4.0
125	3	22.8	5.71	25.3	127	9.2
250	1	22.8	5.73	25.4	127	9.2
250	2	22.8	5.66	25.1	126	8.3
250	3	22.8	5.71	25.3	127	9.2
500	1	22.8	5.75	25.5	128	10.0
500	2	22.8	5.56	24.6	123	5.7
500	3	22.8	5.36	23.7	119	2.3
1000	1	22.8	5.45	24.1	121	4.0
1000	2	22.8	5.29	23.4	117	0.6
1000	3	22.8	5.48	24.3	122	4.9
c 1000	1	22.8	4.17	18.5	92	-
c 1000	2	22.8	4.36	19.3	97	-
c 1000	3	22.8	4.26	18.9	94	-

Results of the nitrate determination at the start of exposure (day 7)

a) water content of 14.2 g/100g Dw;

b) Inhibition is indicated in negative values and stimulation is in positive values;

c) test assay without lucerne meal.

Test concentration [mg/kg Dw] nominal	Replicate	Test mixture for analyses	Eluate	Eluate	Test assay	b Change
			nitrate- nitrogen	calculated nitrate	calculated nitrate
		[g] a	[mg/L]	[mg/L]	[mg/kg Dw]	[%]
Blank control	1	22.8	5.19	23.0	115	-
Blank control	2	22.8	4.65	20.6	103	-
Blank control	3	22.8	5.09	22.5	113	-
62.5	1	22.8	5.10	22.6	113	2.4
62.5	2	22.8	5.24	23.2	116	5.1
62.5	3	22.8	5.07	22.5	112	1.5
125	1	22.8	5.39	23.9	120	8.8
125	2	22.8	5.15	22.8	114	3.3
125	3	22.8	5.26	23.3	117	6.0
250	1	22.8	4.95	21.9	110	-0.3
250	2	22.8	5.19	23.0	115	4.2
250	3	22.8	5.16	22.9	114	3.3
500	1	22.8	5.60	24.8	124	12.4
500	2	22.8	4.95	21.9	110	-0.3
500	3	22.8	5.24	23.2	116	5.1
1000	1	22.8	4.47	19.8	99	-10.3
1000	2	22.8	4.73	20.9	105	-4.8
1000	3	22.8	4.40	19.5	98	-11.2
c 1000	1	22.8	3.74	16.6	83	-9.8d
c 1000	2	22.8	3.94	17.4	87	-5.4d
c 1000	3	22.8	3.75	16.6	83	-9.8d

Results of the nitrate determination at the start of exposure (day 14)

a) water content of 14.2 g/100g Dw;

b) Inhibition is indicated in negative values and stimulation is in positive values;

c) test assay without lucerne meal.

Test concentration [mg/kg Dw] nominal	Replicate	Test mixture for analyses	Eluate	Eluate	Test assay	b Change
			nitrate- nitrogen	calculated nitrate	calculated nitrate
		[g] a	[mg/L]	[mg/L]	[mg/kg Dw]	[%]
Blank control	1	22.8	6.92	30.6	153	-
Blank control	2	22.8	6.79	30.1	151	-
Blank control	3	22.8	6.96	30.8	154	-
62.5	1	22.8	8.39	37.2	186	21.8
62.5	2	22.8	8.47	37.5	188	23.1
62.5	3	22.8	8.64	38.3	192	25.8
125	1	22.8	7.69	34.1	171	12.0
125	2	22.8	7.79	34.5	173	13.3
125	3	22.8	7.78	34.5	173	13.3
250	1	22.8	7.69	34.1	171	12.0
250	2	22.8	8.56	37.9	190	24.5
250	3	22.8	7.58	33.6	168	10.0
500	1	22.8	7.99	35.4	177	15.9
500	2	22.8	7.81	34.6	173	13.3
500	3	22.8	8.10	35.9	180	17.9
1000	1	22.8	7.17	31.8	159	4.1
1000	2	22.8	7.15	31.7	159	4.1
1000	3	22.8	7.38	32.7	164	7.4
c 1000	1	22.8	4.24	18.8	94	2.2d
c 1000	2	22.8	4.38	19.4	97	5.4d
c 1000	3	22.8	4.66	20.6	103	12.0d

Results of the nitrate determination at the start of exposure (day 28)

a) water content of 14.2 g/100g Dw;

b) Inhibition is indicated in negative values and stimulation is in positive values;

c) test assay without lucerne meal.

Test concentration [mg/kg Dw] nominal	Replicate	Test mixture for analyses	Eluate	Eluate	Test assay	b Change
			nitrate- nitrogen	calculated nitrate	calculated nitrate
		[g] a	[mg/L]	[mg/L]	[mg/kg Dw]	[%]
Blank control	1	22.8	9.94	44.0	220	-
Blank control	2	22.8	10.00	44.3	222	-
Blank control	3	22.8	9.66	42.8	214	-
62.5	1	22.8	12.50	55.4	277	26.7
62.5	2	22.8	12.60	55.8	279	27.6
62.5	3	22.8	12.80	56.7	284	29.9
125	1	22.8	11.60	51.4	257	17.5
125	2	22.8	11.30	50.0	251	14.8
125	3	22.8	11.70	51.8	259	18.4
250	1	22.8	11.40	50.5	253	15.7
250	2	22.8	11.30	50.0	251	14.8
250	3	22.8	11.20	49.6	248	13.4
500	1	22.8	11.30	50.0	251	14.8
500	2	22.8	11.20	49.6	248	13.4
500	3	22.8	11.20	49.6	248	13.4
1000	1	22.8	10.60	46.9	235	7.5
1000	2	22.8	10.40	46.1	231	5.6
1000	3	22.8	10.80	47.8	239	9.3
c 1000	1	22.8	5.47	24.2	121	31.5d
c 1000	2	22.8	5.69	25.2	126	37.0d
c 1000	3	22.7	5.62	24.9	125	35.9d

Validity criteria fulfilled:

yes

Remarks:

The results in this study are consistent with all relevant validity criteria and the test is valid according to the guidelines of this study.

Conclusions:

EC 10 >= 1000 mg/kg DW
EC 25 >= 1000 mg/kg DW
EC 50 >= 1000 mg/kg DW

Executive summary:

At the highest test concentration of 1000 mg/kg soil (dw), no negative impairment on nitrogen transformation was observed. In contrast, the application of test substance caused concentration-dependent stimulation effects, namely concentration dependent increased nitrate values at day 28 and increased nitrogen transformation rates (difference of nitrate content between day 28 and day 0).

Therefore, the EC10, EC25 and EC50 values were estimated to be likely >1000 mg/kg Dw while no inhibition was found compared to the controls.

Because the degree of inhibition at 1000 mg/kg Dw was less than 25 % at the end of exposure according to the OECD guideline 216 the test substance can be evaluated as having no long-term influence on nitrogen transformation in soils.

Description of key information

Te endpoint is evaluated by read across of a very similar substance: No toxic effects have been observed in studies with the test item (read across) in soil living microorganisms.

EC 10 >= 1000 mg/kg DW

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:

1 000 mg/kg soil dw

Additional information

In a 28-day toxicity study on the soil nitrogen transformation activity according to OECD 216, natural soil was exposed to the test substance at nominal concentrations of 0 (blank control with solvent), 62.5 mg/kg, 125 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg (dry weight). Lucerne meal was used as the additional nitrogen source. Nitrate formed during microbial processes was determined at the start of exposure (Day 0), after 7, 14 and at the end of exposure (Day 28) and were compared to control assays of the corresponding days.

There is an overall stimulation of organic nitrogen breakdown in the 62.5mg /kg dry soil weight, which is reflected in the results by increased levels of the end product of mineralization and nitrification, nitrate, by Day 28.

At the highest test concentration of 1000 mg/kg soil (dw), no negative impairment on nitrogen transformation was observed. In contrast, the application of test substance caused concentration-dependent stimulation effects, namely concentration dependent increased nitrate values at day 28 and increased nitrogen transformation rates (difference of nitrate content between day 28 and day 0).

There were no morphological observations made during the incubation and the soil remained visually unchanged during exposure period.

Therefore, the EC10, EC25 and EC50 values were estimated to be likely >1000 mg/kg Dw while no inhibition was found compared to the controls.

Because the degree of inhibition at 1000 mg/kg Dw was less than 25 % at the end of exposure according to the OECD guideline 216, the test substance can be evaluated as having no long-term influence on nitrogen transformation in soils.