(3E)-1,7,7-trimethyl-3-(4-methylbenzylidene)bicyclo[2.2.1]heptan-2-one

Toxicity to soil microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-05-07 until 2018-12-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Version / remarks:

Year 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

yes

Details on preparation and application of test substrate:

AMENDMENT OF SOIL
- Type of organic substrate: Freshly sampled and 2mm sieved soil from the LUFA Speyer, standard soil type 2.3, silty sand (Sand content: 57.3%; pH: 5.9; organic carbon content: 0.66%)

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: Application solution (20 mL) was applied dropwise to quartz sand (40g) and carefully mixed with a mortar. After evaporation of the solvent the quartz sand-test item mixture was mixed with a soil batch of 500 g dry soil per treatment.

VEHICLE:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetone
- Evaporation of vehicle before use: yes

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

20 +/- 2°C

Moisture:

Soil water content was adjusted to about 49.32 % MWC and was was controlled weekly by weighing.

Details on test conditions:

TEST SYSTEM
- Test container (type, material, size): Glass incubation flasks, 1 L, stoppered with cotton wool plugs
- Amount of soil: 150g (dw)
- No. of replicates per concentration: 2
- No. of replicates per control: 2
- No. of replicates per vehicle control: 2

SOIL INCUBATION
- Method: The soil arrived on 26-Apr-2018 (DAT -54) at IES Ltd., was stored in the dark under aerobic conditions at approx. 4 °C for 28 days and then pre-moistened to approx. 40 % MWHC. Afterwards, the pre-moistened soil was acclimatised to test conditions (dark, approx. 20 °C) for 26 days until test start on 19-Jun-2018 (DAT 0). Prior to application, the soil moisture content was adjusted to 45 % MWHC.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): LUFA Speyer 2.3
- Vegetation cover: No cultivation 2014 - 2018
- Treatments with pesticides or fertilizers: No fertilization 2017 – 2018 and 2015 – 2016; 2014 3 x 3500 kg CaO/ha, 1463 kg MgO/ha; No pesticides 2014 - 2018
- Depth of sampling: approx. 20 cm
- Soil type: Silty sand
- % sand: 57.3%
- pH (in water): 5.9
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): 0.08 %
- Maximum water holding capacity (in % dry weigth): 34.9
- Cation exchange capacity (mmol/kg): 7.3
- Initial microbial biomass as % of total organic C: 3.23 (The microbial biomass of the test soil was measured by the fumigation method. The mean values of soluble total organic carbon of the fumigated soil and not fumigated soil samples were 137.5 and 56.4 mg/kg dry soil, respectively, resulting in the calculation of a microbial biomass of 213.3 mg/kg dry soil)

The soil for the nitrification measurements was amended with powdered lucerne meal as a source of organic nitrogen before application of the test item. The lucerne meal had contents of 41.9 % carbon and 2.65 % nitrogen resulting in a C:N ratio of approx. 15.8:1 and was added to the soil in amounts of approx. 5 g/kg dry soil.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Nitrification was determined at DAT 0 and 28

VEHICLE CONTROL PERFORMED: yes

Nominal and measured concentrations:

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

Reference substance (positive control):

yes

Remarks:

Nitrapyrin PESTANAL (CAS 1929-82-4)

Key result

Duration:

28 d

Dose descriptor:

NOEC

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:

The daily soil nitrate formation rate was calculated from the nitrate concentrations at DAT 0 and 28 (Table 5). The daily nitrate formation rate in the control and solvent control was -0.23 and -0.84 mg/kg dry soil/day, respectively, and differed statistically significantly from each other (Student t-test, two-sided, α = 0.05). This difference was considered to be the consequence of calculations based on different starting nitrate concentrations at Day 0 (see section 6.2.3). During the 28 days of the study, the soil microflora in the control and solvent control had sufficient time to equilibrate and produced comparable values of nitrate concentration at DAT 28 (see section 6.2.4). Since the solvent control and the test item treatments were treated with the same solvent, the solvent control was used for further statistical comparisons with the reference item treatment as well as with the test item treatments.
In the test item treatments, mean nitrate formation rate was in the range of -0.92 to 1.38 mg nitrate/kg dry soil/day without a concentration-effect relationship and was not statistically significantly smaller compared to the solvent control (Dunnett t-test, one-sided smaller, α = 0.05). A decrease of soil nitrate concentration between Day 0 and 28 can occur when the microbial community of nitrifiers and denitrifiers change during the course of the study and was found in this study for the majority of the treatments. This decrease was not considered to be caused by the test item since no concentration-effect relationship on nitrate formation rate was observed.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
The mean nitrate formation rate at DAT 28 in the reference item treatment was 94 % lower than in the solvent control and differed statistically significantly from the solvent control (Student t-test, one-sided smaller, α = 0.05; due to the outlier test after Hampel, replicate 3 of the reference item treatment was excluded). The statistically significantly decreased nitrate formation rate in the reference item treatment was the expected effect of the reference item in order to demonstrate that the used test system was suitable to detect potential effects of the test item on the nitrification potential of the exposed soil microflora.

Results nitrate concentration

Treatment	Incu-bation time [days]	Nitrate concentration [mg NO3^-/ kg dry soil]							Stat.
		Replicates						% deviation from control
		a	b	c	Mean	SD	CV
Control	0	56,5	55,8	56,0	56,1	0,4	1	-21	s.^a
	28	49,3	48,5	51,5	49,8	1,6	3	5	n.s.^a
Solvent Control Aceton	0	71,3	71,0	70,0	70,8	0,7	1	0	-
	28	45,5	47,5	49,0	47,3	1,8	4	0	-
Neo Heliopan MBC: 62.5 mg/kg dry soil	0	62,3	53,0	53,5	56,3	5,2	9	-20	n.s.
	28	93,5	92,3	98,8	94,8	3,4	4	100	n.s.
Neo Heliopan MBC: 125 mg/kg dry soil	0	55,0	50,3	67,3	57,5	8,8	15	-19	n.s.
	28	38,5	24,3	32,8	31,8	7,2	23	-33	s.
Neo Heliopan MBC: 250 mg/kg dry soil	0	49,5	47,5	47,8	48,3	1,1	2	-32	s.
	28	67,3	64,5	60,5	64,1	3,4	5	35	n.s.
Neo Heliopan MBC: 500 mg/kg dry soil	0	87,0	87,0	93,3	89,1	3,6	4	26	s.
	28	70,3	67,0	80,5	72,6	7,0	10	53	n.s.
Neo Heliopan MBC: 1000 mg/kg dry soil	0	66,8	91,3	71,8	76,6	12,9	17	8	n.s.
	28	64,3	78,5	85,5	76,1	10,8	14	61	n.s.
6.7 mg Nitrapyrin/kg dry soil^a	0	80,5	97,0	72,0	88,8	11,7	13	25	n.s.^b
	28	33,0	53,8	57,8	43,4	14,7	34	-8	n.s.^c

 

Results nitrate formation rate

Treatment	Incu-bation time [days]	Nitrate formation rate [mg NO3^-/ kg dry soil/day]							Stat.
		Replicates						% deviation from control
		a	b	c	Mean	SD	CV
Control	28	-0,26	-0,26	-0,16	-0,23	0,06	-25	-73	s.^a
Solvent Control Aceton	28	-0,92	-0,84	-0,75	-0,84	0,08	-10	0	-
Neo Heliopan MBC: 62.5 mg/kg dry soil	28	1,12	1,40	1,62	1,38	0,25	18	-265	n.s.
Neo Heliopan MBC: 125 mg/kg dry soil	28	-0,59	-0,93	-1,23	-0,92	0,32	-35	10	n.s.
Neo Heliopan MBC: 250 mg/kg dry soil	28	0,63	0,61	0,46	0,57	0,10	17	-168	n.s.
Neo Heliopan MBC: 500 mg/kg dry soil	28	-0,60	-0,71	-0,46	-0,59	0,13	-22	-30	n.s.
Neo Heliopan MBC: 1000 mg/kg dry soil	28	-0,09	-0,46	0,49	-0,02	0,48	-2673	-98	n.s.
6.7 mg Nitrapyrin/kg dry soil^a	28	-1,70	-1,54	-0,51	-1,62	0,11	-7	94	s.^b

Validity criteria fulfilled:

yes

Conclusions:

The NOEC for nitrate concentration and formation were >= 1000 mg/kg dry soil.

Executive summary:

In a valid, reliable and conclusive study according to OECD guideline 216 (2000), soil microorganisms were exposed to the test item in natural soil for 28 days. The NOEC and LOEC for nitrate concentration and nitrate formation at Day 28 were ≥ 1000 and > 1000 mg/kg dry soil, respectively.

Description of key information

The NOEC (28 days) for nitrate concentration and formation was found to be >= 1000 mg/kg dry soil in an OECD 216 test with soil microorganisms.

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:

1 000 mg/kg soil dw