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

Toxicity to terrestrial arthropods

Link to relevant study record(s)

Reference

Endpoint:

toxicity to terrestrial arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-05-11 - 2018-10-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 232 (Collembolan Reproduction Test in Soil)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Application method:

soil

Analytical monitoring:

yes

Remarks:

Application solutions were analysed

Details on sampling:

Samples from the freshly prepared application solutions were deep-frozen directly after application and transferred to the analytical department for analysis after the end of the biological phase.
Duplicate samples of every test item concentration as well as of the solvent control treatment were taken from the application solutions. The samples were stored at approximately - 20 °C.
One of the duplicate samples was analysed using HPLC/UV while the other one was kept as a spare sample.

Vehicle:

yes

Remarks:

Acetone

Details on preparation and application of test substrate:

For each treatment, 150 g of fresh soil (taking into account that 50 g sand were needed for the preparation of the test-item sand mixture) with a remaining moisture of 0.38 % (corresponding to 199.24 g dry soil with 0.0 % moisture) was humidified with 50 mL ultrapure water in two portions: 20 mL were added four days before application by pre-wetting the soil substrate (without 50 g sand needed for application), during application the remaining 30 mL ultrapure water were mixed in the pre-wetted soil in parallel with 50 g sand (untreated for the control, after evaporation of the solvent for the solvent control, after evaporation of the solvent from the test item-solvent mixture in the test item treatments).
For the preparation of the application solution of the highest test item concentration (solution 1) the weighed amount of 664.15 mg of the test item (target: 664.1 mg) was dispersed in 100 mL acetone under intense stirring for 15 minutes.
For the preparation of the test item-sand mixtures, no solvent was added to 50 g sand (for the control), 30 mL acetone was added to 50 g sand (for the solvent control) and 30 mL application solution was added to 50 g sand (for each of the test item treatments). The treated sand was kept under a hood for evaporation for 4.5 hours.
During application, the amounts of 50 g sand (untreated for the control, after evaporation of the solvent for the solvent control, after avaporation of the solvent from the test item-solvent mixture for the test item treatments) were mixed with 150 g pre-wetted soil substrate (for the control), 2 x 150 g pre-wetted soil substrate (for the solvent control) and 150 g pre-wetted soil substrate (for each of the test item treatments). In parallel, 30 mL purifed water (for the control), 2 x 30 mL purifed water (for the solvent control) or 30 mL purifed water were added to the treated soil substrate to reach the water content of 40 to 60 % of the maximum water holding capacity (MWHC). The mixing period was approx. 5 minutes using a laboratory mixer.

Test organisms (species):

Folsomia candida

Animal group:

Collembola (soil-dwelling springtail)

Details on test organisms:

The study was conducted with the springtail species Folsomia candida (Collembola: Isotomidae) from a culture maintained at IES Ltd. Synchronized 12 day-old F. candida juveniles were used for the study. The synchronized individuals were kept at room temperature in breeding units under total darkness and fed with granulated dry baker’s yeast.

Study type:

laboratory study

Limit test:

no

Total exposure duration:

28 d

Test temperature:

20 +/- 2°C

pH (if soil or dung study):

6.1 - 6.4 (start)
5.9 - 6.5 (end)

Humidity:

At test initiation, water content was 21.68 – 24.43 % (corresponding to 41.1 – 46.3 % of the MWHC). At test termination, water content was 20.69 – 24.53 % (corresponding to 39.2 – 46.5 % of the MWHC).

Photoperiod and lighting:

16:8 h light:dark

Details on test conditions:

TEST SYSTEM
- Test container (material, size): glass containers (8.5 cm height x 4 cm diameter)
- Amount of soil or dung or substrate: 30g (dry weight)
- Depth of dung (cm): 2.4 cm
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 4
- No. of replicates per vehicle control: 8

SOURCE AND PROPERTIES OF SUBSTRATE (if soil or dung)
The artificial soil substrate was prepared according to the test guideline procedures described in OECD Guideline 232, with the following constituents (final percentage distribution on
dry weight basis):
· 5 % sphagnum peat (air dried and finely ground, with no visible plant remains),
· 20 % kaolin clay (kaolinite content > 30 %),
· approximately 74 % fine quartz-sand, and
· ≤ 1 % calcium carbonate (CaCO3, pulverised, analytical grade) to obtain a pH of 6.0 ±
0.5.
- Maximum water holding capacity (in % dry weight): 52.74%

OTHER TEST CONDITIONS
- Photoperiod: 16:8
- Light intensity: 572 to 707 lux at test initiation

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality, Reproduction (after 28 days)

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Test concentrations: Nominal 16.3, 29.4, 52.9, 95.3, 171, 309, 556 and 1000 mg/kg dry soil

Nominal and measured concentrations:

Nominal 16.3, 29.4, 52.9, 95.3, 171, 309, 556 and 1000 mg/kg dry soil
The concentration of the test item in the application solutions was analytically determined to be 91 – 98 % of nominal.

Reference substance (positive control):

yes

Remarks:

Boric Acid (IES 20170193, August 2017)

Key result

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

28.2 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

29.4 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

52.9 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

28 d

Dose descriptor:

LC10

Effect conc.:

32.5 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

29.4 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

LC50

Effect conc.:

56.8 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

52.9 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

- Mortality at end of exposure period: Control: 2.5%; Solvent control: 6.25% (no significant difference, controls were pooled); Mortality in the test item treatments was 2.5 and 5.0 % at the two lowest test item treatments of 16.3 and 29.4 mg/kg dry soil and increased then in a concentration-effect relationship up to 100 % from 171 mg/kg dry soil on. Mortality from 52.9 mg/kg dry soil on was statistically significantly greater compared to the control (step-down Cochran-Armitage test, one-sided greater, α = 0.05).

-Reproduction:
The mean number of juveniles in the control and the solvent control was 176 and 157, respectively, and the corresponding coefficient of variation of the number of juveniles was 13 and 21 %, respectively. Therefore, both validity criteria for reproduction in the control and the solvent control (i.e. mean number of juveniles ≥ 100 and coefficient of variation < 30 %) were fulfilled. Mean reproduction in the control and the solvent control did not differ statistically significantly from each other (Student-t test, two-sided, α = 0.05). Therefore, the controls were pooled in order to increase the statistical significance of the following test.
Mean reproduction in the test item treatments was 109 and 99 % of the control at the two lowest test item treatments of 16.3 and 29.4 mg/kg dry soil and decreased then in a concentrationeffect relationship up to 0 % of the control from 171 mg/kg dry soil on. Mean reproduction from 52.9 mg/kg dry soil on was statistically significantly smaller compared to the control (Welch-t test for inhomogeneous variances, one-sided greater, α = 0.05).

Results with reference substance (positive control):

EC50 for reproduction was 116 mg/kg dry soil (95 % confidence interval 101 - 134 mg/kg dry soil (August 2017).
This value corresponded with the recommendations given in the OECD testing guideline No. 232 (2016, [1]) where Boric Acid should reduce reproduction by 50 % at about 100 mg/kg dry weight soil. Therefore, the used test system was suitable to show effects of the reference item on reproduction of Folsomia candida in the expected range of test concentrations.

Mortality of Folsomia candida adults after 28 days

Treatment group	Nominal concentration (mg /kg dry soil)	Rep.	N°. ofF. candidajuveniles introduced on day 0	N°. ofF. candidaadults found on day 28	N°. of deadF. candidaadults on day 28	Mean mortality (%)			Stat.
						Mean	±	SD
Control	-	1	10	10	0	2,5	±	5,0	n.s.^a
		2	10	9	1
		3	10	10	0
		4	10	10	0
Solvent control	-	1	10	9	1	6,25	±	7,4	n.s.^a
		2	10	8	2
		3	10	9	1
		4	10	9	1
		5	10	10	0
		6	10	10	0
		7	10	10	0
		8	10	10	0
Pooled control	-	1-12	120	114	6	5,0	±	6,7	-
Neo Heliopan MBC	16,3	1	10	10	0	2,5	±	5,0	n.s.
		2	10	10	0
		3	10	9	1
		4	10	10	0
	29,4	1	10	10	0	5,0	±	10,0	n.s.
		2	10	10	0
		3	10	8	2
		4	10	10	0
	52,9	1	10	9	1	35	±	19	s.
		2	10	5	5
		3	10	5	5
		4	10	7	3
	95,3	1	10	3	7	92,5	±	15,0	s.
		2	10	0	10
		3	10	0	10
		4	10	0	10
	171	1	10	0	10	100	±	0,0	s.
		2	10	0	10
		3	10	0	10
		4	10	0	10
	309	1	10	0	10	100	±	0,0	s.
		2	10	0	10
		3	10	0	10
		4	10	0	10
	556	1	10	0	10	100	±	0,0	s.
		2	10	0	10
		3	10	0	10
		4	10	0	10
	1000	1	10	0	10	100	±	0,0	s.
		2	10	0	10
		3	10	0	10
		4	10	0	10

Note: Number of 10 adults F. candida inserted per replicate

Stat: Statstical analysis of differences between the control and the solvent control using the Binomial Fisher Exact test (two-sided, α = 0.05) as well as differences betrween the test item treatments and the pooled control using the Step.down Cochran Armitage test (one-sided smaller, α = 0.05)

n.s.a = not statistically significant (control versus solvent control)

n.s.: = not statistically significant (test item treatments versus pooled control)

s. = Statistically significant (test item treatments versus pooled control)

Reproduction of Folsomia candida adults after 28 days

Treatment group	Nominal concen-tration (mg/kg dry soil)	Rep.	N°. ofF. candidaon day 28
			Adults per replicate	Juveniles per replicate	Mean	±	SD	CV	% of pooled control	Stat.
Control	-	1	10	149	176	±	24	13	108	n.s.^a
		2	9	205
		3	10	168
		4	10	183
Solvent control	-	1	9	152	157	±	33	21	96	n.s.^a
		2	8	111
		3	9	151
		4	9	128
		5	10	208
		6	10	143
		7	10	159
		8	10	202
Pooled control	-	1-12	114	163	163	±	31	19	-	-
Neo Heliopan MBC	16,3	1	10	187	178	±	23	13	109	n.s.
		2	0	206
		3	0	163
		4	0	156
	29,4	1	10	139	161	±	49	30	99	n.s.
		2	10	179
		3	8	107
		4	10	220
	52,9	1	9	95	59	±	29	50	36	s.
		2	5	26
		3	5	65
		4	7	48
	95,3	1	3	11	3	±	6	200	2	s.
		2	0	0
		3	0	0
		4	0	0
	171	1	0	0	0,25	±	1	200	0	s.
		2	0	0
		3	0	0
		4	0	1
	309	1	0	0	0	±	0	n.a.	0	s.
		2	0	0
		3	0	0
		4	0	0
	556	1	0	0	0	±	0	n.a.	0	s.
		2	0	0
		3	0	0
		4	0	0
	1000	1	0	0	0	±	0	n.a.	0	s.
		2	0	0
		3	0	0
		4	0	0

Note: Number of 10 adults F. candida inserted per replicate

Stat: Statstical analysis of differences between the control and the solvent control using the Student t-test (two-sided, α = 0.05) as well as differences betrween the test item treatments and the pooled control using the Welch t.test for inhomogeneous variances (one-sided smaller, α = 0.05)

n.s.a = not statistically significant (control versus solvent control)

n.s.: = not statistically significant (test item treatments versus pooled control)

s. = Statistically significant (test item treatments versus pooled control)

Validity criteria fulfilled:

yes

Conclusions:

The EC10 for reproduction was 28.2 mg/kg dry soil.

Executive summary:

In a valid, reliable and conclusive study, the effects of the test item on mortality and reproduction of the springtail species Folsomia candida (Collembola: Isotomidae) were tested for 28 days according to the OECD testing guideline No. 232 (2016).

The concentration of the test item in the application solutions was analytically determined to be 91 – 98 % of nominal. Therefore, all results were related to nominal test concentrations.

The most sensitive endpoint is reproduction with an EC10 = 28.2. mg/kg dw. The NOEC and LOEC for mortality and reproduction were 29.4 and 52.9 mg/kg dry soil, respectively.

Description of key information

The EC10 (28 days) for reproduction was found to be 28.2 mg/kg dry soil in an OECD 232 Collembolan Reproduction test in soil with Folsomia candida.

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for soil dwelling arthropods:

28.2 mg/kg soil dw