Reaction mass of (3aR,5aS,9aS,9bR)-3a,6,6,9a-Tetramethyldodecahydronaphtho[2,1-b]furan and (3aS,5aR,9aR,9bS)-3a,6,6,9a-Tetramethyldodecahydronaphtho[2,1-b]furan

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 25 February 2021 to 23 April 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: SANCO/3029/99 rev.4, Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A, Section 4) and Annex III (part A, Section 5) of Directive 91/414 (11/07/00)

Version / remarks:

rev 4

Qualifier:

according to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

Version / remarks:

2016

Deviations:

yes

Remarks:

Food was provided on day 0 instead of Day 1 At the end, the soil moisture deviated by more than 10% from the initial value in all treatments The room temperature decreased to minimum 17°C for about 32h.

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

(incl. QA statement) inspected on 12 - 15 November 2019 / signed on 27 August 2020

Specific details on test material used for the study:

Appearance : White solid
Water solubility : 1.88 mg/L at 20 °C (OECD 105)
Solubility in solvents : Not soluble in acetone, n-hexane and methanol
Storage condition : dry area, protect from light, in refrigerator (2-8°C), store under nitrogen in a closed container after first opening

Analytical monitoring:

yes

Remarks:

GC-MS

Details on sampling:

Sampling : On day 0 and day 28 the amount of test item was analytically verified via GC-MS for the control and all test item concentrations using mixed samples of all replicates, each.

Vehicle:

no

Details on preparation and application of test substrate:

Application into the artificial soil by mixing.
The test item was treated with a mortar. The respective test item amount was weighed out for each test item concentration and mixed thoroughly with quartz sand (10g per replicate). The spiked quartz sand was added to the artificial soil. Demineralised water was added to adjust the humidity of the artificial soil to a moisture of 40 – 60 % of the maximum water holding capacity. Subsequently, the test medium will be thoroughly mixed to ensure a homogenous distribution and about 610 g soil dry weight will be filled into each test vessel.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

Source : Breeding stock culture maintained at the test facility
Breeding : Organisms of the species Eisenia fetida are bred at the test facility in covered plastic vessels containing potting compost.
Synchronisation of the earthworm population: Earthworms of homogeneous age and body weight were used in the definitive test. Synchronisation of the population was achieved by placing adult earthworms into breeding boxes and removing the adults after 4 weeks. Offspring from the remaining cocoons reached the adult age after 2 months at the earliest.
Feeding : During breeding, the earthworms are fed with a litter of dried stinging nettle leaves and porridge oats. A sufficient amount of the food was provided depending on the feeding rate and the density of the earthworm population in the vessels.

Study type:

laboratory study

Substrate type:

artificial soil

Remarks:

- 5 % peat, air-dried and finely ground - 20 % kaolin, kaolinite content > 30 % - 74 % air-dried quartz sand (sand with > 50 % particle size of 0.05 - 0.2 mm) - 0.13 % calcium carbonate (CaCO3) to achieve a pH of 6.0  0.5

Limit test:

no

Total exposure duration:

8 wk

Test temperature:

Nominal : 20+/- 2 °C
Actual : 17 - 22 °C
The room temperature decreased to minimum 17 °C for about 32 hours. This deviation is considered to have no impact on quality and integrity of the study.

pH:

The initial pH-value of the artificial soil was 6.50 (measured on day -6 before start of the
exposure).
The pH in the control was 6.38 at test start and 6.22 at test end.
The pH in test vessels was between 6.09 and 6.19 at test start and between 6.05 and 6.22 at test end.

Moisture:

The moisture in the control was 17.9 at test start and 22.4 at test end.
The moisture in test vessels was between 17.7 and 18.3 at test start and between 21.4 and 22.7 at test end.
The soil moisture deviated by more than 10 % from the initial value. This deviation is considered to have no impact on quality and integrity of the study.

Details on test conditions:

Test duration : 8 weeks

Number of replicates : 8 replicates per control and 4 replicates per test item concentration

Number and age of the earthworms 10 adult earthworms (with clitellum) aged 2-12 months were used per replicate. The difference of age did not deviate by more than 1month

Body weight (actual): 0.40 to 0.54 g

Adaptation : Earthworms were adapted to the artificial soil to be used for the test two days prior to test start.

Homogeneity of groups : The worms were washed with demineralised water and dried gently on a paper towel. The homogeneity of the population was checked by weighing the earthworms. After confirmation of homogeneity, groups of 10 earthworms (chosen by a randomised procedure) were weighed and introduced into each test vessel.

Test medium Artificial soil consisted of the following components:
- 5 % peat, air-dried and finely ground
- 20 % kaolin, kaolinite content > 30 %
- 74 % air-dried quartz sand (sand with > 50 % particle size of 0.05 - 0.2 mm)
- 0.13 % calcium carbonate (CaCO3) to achieve a pH of 6.0  0.5
The WHCmax, the moisture content and the pH-value of the artificial soil were determined and adjusted. Two days before experimental starting the artificial soil (24000 g) was pre-moistened with 2065 g demineralised water to achieve a moisture content corresponding
to 27 % of the WHCmax of the artificial soil. The moistened soil was kept covered to prevent evaporative water losses prior to the start of exposure.

Test container :Test units containing about 610 g soil dry weight, were round plastic boxes with an inner diameter of 15.0 cm corresponding to a bottom surface area of 177 cm2 and a height of 14 cm. Transparent and perforated lids enable sufficient gas exchange, light input and prevent the test substrate from drying.

Feeding : The earthworms were fed with air-dried cattle manure weekly during the first 4 weeks as presented in Table 4. At test start and at day 28 the food was mixed carefully into the soil, on the other days the food was placed onto the soil surface. No further feeding took place during the remaining 4 weeks of the test.
The cattle manure was delivered by LEHR- UND FORSCHUNGSGUT RUTHE, Schäferberg 1, 31157 Sarstedt, Germany. Manure was obtained from cattle that were not subject to medication or treatment with substances, such as growth promoters, nematicides or similar veterinary products that could adversely affect the worms during the test. The unpasteurized manure was air-dried and finely ground before use.

Moistening : Throughout the test the replicates were weighed weekly to determine water loss via evaporation. Evaporated water was replenished by adding demineralised water to maintain the soil moisture which should not deviate by more than 10% of the initial value at the end of the test. Additional moistening was performed in case the soil appeared too dry

Measurements and observation: At experimental starting and experimental completion, the
pH-value and moisture content of the test medium were determined in each treatment and the control from pooled samples of all replicates. The water content of each test container was checked weekly by weighing. The weight loss was replenished with the appropriate amount of demineralised water. The body weight of the adult earthworms was determined individually on day 0 and day 28 for each replicate. After the first four weeks, the adult earthworms were removed.
Mortality and morphological changes of the adult earthworms were recorded for each replicate.
After a further four weeks, the number of offspring (juveniles) hatched from the cocoons was counted for each test container.

Observation parameters
A) no obvious pathological symptoms
B) no reaction to touching
C) no negative phototactical reaction
D) spontaneous segmentation and separation
E) spasmodic winding
F) yellow excretion from the oral aperture
G) ulcer and skin bleedings
H) dead earthworms

Nominal and measured concentrations:

Nominal test item concentration : 6.25 – 12.5 – 25 – 50 – 100 – 200 mg/kg soil dry weight
Measured concentration Day 0 : 6.96 - 7.67 - 27.9 - 37.3- 93.1- 195 mg/kg soil dry weight
Measured concentration Day 24 : 2.33 - 2.26 - 8.76 - 18.2- 28.3- 44.5 mg/kg soil dry weight

Reference substance (positive control):

yes

Remarks:

A test with Carbendazim is conducted once within twelve months. According to OECD 222 significant effects of Carbendazim should be observed between 1 and 5 mg a.i./kg soil DW. A significant effects was observed at the minimum concentration 0.5 mg a.i./kg

Key result

Duration:

8 wk

Dose descriptor:

LOEC

Effect conc.:

> 200 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality biomass reproduction

Key result

Duration:

8 wk

Dose descriptor:

NOEC

Effect conc.:

>= 200 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality, biomass, reproduction

Key result

Duration:

8 wk

Dose descriptor:

EC10

Effect conc.:

92.2 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

No evident earthworm mortality (< 10%) was observed in the control and all test item concentrations. One dead earthworm was observed in 100 mg and 200 mg after 28 days.
No significant pathological symptoms or changes in the behaviour of adult earthworms were observed in the controls and all test item concentrations.
At test start, the earthworms had individual weights of 0.40 - 0.54 g. There were no statistically significant differences in earthworm body weights in all treatment rates compared to the control.
The reproduction rate (average number of juveniles produced) was 127 in the control and ranged from 106 to 168 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction at all treatment rates compared to the control.
The coefficient of variation calculated for the reproduction of the control was 28.4 % and thus lower than 30 %.

Results with reference substance (positive control):

A test with Carbendazim is conducted once within twelve months. According to OECD 222 significant effects of Carbendazim should be observed between 1 and 5 mg a.i./kg soil DW. A significant effects was observed at the minimum concentration 0.5 mg a.i./kg
Application rate
[mg test item number [%] of
/kg SDW] Replicate of juveniles Mean ± SD CV [%] control Sig.
Control 1 159 114 ± 25.3 22.2 - -
2 98
3 125
4 86
5 131
6 110
7 83
8 120
0.5 1 105 85 ± 17.8 20.9 74.6 Yes
2 67
3 94
4 73
1 1 91 91 ± 25.0 27.5 79.8 Yes
2 109
3 109
4 56
2 1 72 74 ± 10.1 13.6 64.9 Yes
2 60
3 79
4 83
4 1 0 0 ± 0 0.0 0.0 Yes
2 0
3 0
4 0

Reported statistics and error estimates:

Determination of NOEC and LOEC with Dunnett’s Multiple Comparison Test, α = 0.05
Determination of EC10 by sigmoidal dose -response
Biomass and Reproduction - Dunnett’s Multiple Comparison Test
The alpha -value (acceptable probability of incorrectly concluding that there is a difference) was alpha = 0.05.
Determination of EC10 by sigmoidal dose -response
Reproduction The EC10 – value for reproduction was determined runningvSigmoidal dose-response.
Software: The data presented in the tables of the report were computergenerated and have been rounded for presentation. Thus, manual re-calculation of the data based on the results presented in this report may result in minor deviations from these figures.
Calculations were carried out using the following software:
- Excel, MICROSOFT
- GraphPad Prism version 5.03, GRAPHPAD SOFTWARE INC.

Mortality of Adult Earthworms in [%] after 28 Days of Exposure

Application rate [mg test item/kg SDW]	Replicate
	1	2	3	4	5	6	7	8	Mean
Control	0	0	0	0	0	0	0	0	0
6.25	0	0	0	0	-				0
12.5	0	0	0	0					0
25	0	0	0	0					0
50	0	0	0	0					0
100	0	0	10	0					2.5
200	0	0	10	0					2.5

 

Earthworm Behaviour and Pathological Symptoms after 28 Days of Exposure

Application rate [mg test item/kg SDW]		Replicate
	Observation parameter	1	2	3	4	5	6	7	8
Control	A	10/10	10/10	10/10	10/10	10/10	10/10	10/10	10/10
6.25	A	10/10	10/10	10/10	10/10	-
12.5	A	10/10	10/10	10/10	10/10
25	A	10/10	10/10	10/10	10/10
50	A	10/10	10/10	10/10	10/10
100	A H	10/10 -	10/10 -	9/10 1/10	10/10 -
200	A H	10/10 -	10/10 -	9/10 1/10	10/10 -

A = no obvious pathological symptoms H = earthworm dead - = no observation

 

Body Weight Changes of the Adult Earthworms

Application rate [mg test item/kg SDW]	Replicate	Mean body weights per replicate		Mean body weight change of earthworms
		Test start	28 days	Per replicate		Mean ± SD		Sig.
		[g]	[g]	[g]	[%]	[g]	[%]
Control	1	0.46	0.52	0.06	13.0	0.08 ± 0.03	17.3 ± 5.91	-
	2	0.45	0.56	0.11	24.4
	3	0.46	0.53	0.07	15.2
	4	0.45	0.52	0.07	15.6
	5	0.46	0.55	0.09	19.6
	6	0.45	0.57	0.12	26.7
	7	0.46	0.53	0.07	15.2
	8	0.45	0.49	0.04	8.9
6.25	1	0.45	0.59	0.14	31.1	0.11 ± 0.03	23.7 ± 6.83	NO
	2	0.46	0.53	0.07	15.2
	3	0.45	0.57	0.12	26.7
	4	0.46	0.56	0.10	21.7
12.5	1	0.45	0.53	0.08	17.8	0.09 ± 0.02	18.7 ± 5.08	No
	2	0.46	0.53	0.07	15.2
	3	0.45	0.52	0.07	15.6
	4	0.46	0.58	0.12	26.1
25	1	0.46	0.55	0.09	19.6	0.09 ± 0.01	20.3 ± 2.03	No
	2	0.45	0.53	0.08	17.8
	3	0.46	0.56	0.10	21.7
	4	0.45	0.55	0.10	22.2
50	1	0.46	0.57	0.11	23.9	0.10 ± 0.01	22.0 ± 2.53	No
	2	0.45	0.56	0.11	24.4
	3	0.46	0.55	0.09	19.6
	4	0.45	0.54	0.09	20.0
100	1	0.46	0.61	0.15	32.6	0.11 ± 0.03	24.1 ± 5.73	No
	2	0.45	0.54	0.09	20.0
	3	0.46	0.56	0.10	21.7
	4	0.45	0.55	0.10	22.2
200	1	0.46	0.59	0.13	28.3	0.09 ± 0.05	19.7 ± 9.79	No
	2	0.45	0.48	0.03	6.7
	3	0.46	0.58	0.12	26.1
	4	0.45	0.53	0.08	17.8

 

SD = Standard deviation

Sig. = Statistical Significance (Dunnett’s Multiple Comparison Test, α = 0.05) compared to control

Reproduction Rate (Number of Juveniles after 8 Weeks)

Application rate [mg test item/kg SDW]	Replicate	number of juveniles	Mean ± SD	CV [%]	[%] of control	Sig.
Control	1	87	127 ± 36.0	28.4	-	-
	2	128
	3	150
	4	69
	5	135
	6	114
	7	181
	8	148
6.25	1	132	144 ± 25.3	17.5	113	No
	2	172
	3	115
	4	156
12.5	1	102	157 ± 36.9	23.5	124	No
	2	177
	3	175
	4	175
25	1	142	168 ± 30.7	18.3	132	No
	2	178
	3	145
	4	207
50	1	147	144 ± 18.1	12.6	113	No
	2	142
	3	165
	4	121
100	1	134	106 ± 41.1	38.8	83.5	No
	2	61
	3	82
	4	147
200	1	119	115 ± 20.9	18.1	90.6	No
	2	123
	3	85
	4	133

 

SD = Standard deviation CV = Coefficient of variation

Sig. = Statistical Significance (Dunnett’s Multiple Comparison Test, α = 0.05) compared to the control

Validity criteria fulfilled:

yes

Remarks:

Adult mortality in the control group after the first four weeks of the definitive test : 0 % < 10% Average number of juveniles in the control group:127 > 30 Coefficient of variation calculated for the reproduction of the control group : 28.4 % < 30%

Conclusions:

Under the conditions of this study, neither evident earthworm mortalities (< 10%) nor evident
pathological symptoms or changes in the behaviour of adult earthworms were observed in the
control or all test item concentrations. There were no statistically significant differences in
earthworm body weights in all test item concentrations compared to the control. After a further
four weeks, the reproduction rate (average number of juveniles produced) was 127 in the control
and ranged from 106 to 168 in the test item treatment rates. There were no statistically significant
differences in earthworm reproduction in all treatment rates compared to the control.

Executive summary:

Effects of registered substance on mortality, biomass and the reproductive potential of the earthworm species Eisenia fetida (Annelida, Lumbricidae) were determined according to OECD 222 (2016).

The study was conducted under static conditions over 8 weeks with the test item concentrations 6.25 - 12.5 – 25 – 50 – 100 – 200 mg test item/kg SDW. Each application rate was mixed into artificial soil containing 5 % peat. A control including untreated artificial soil was tested under the same conditions as the test item treatments. 80 test organisms were inserted into 8 control replicates. 40 test organisms were divided into 4 replicates for each test item treatment (10 earthworms per replicate). They had an individual body weight between 0.40 and 0.54 g at experimental starting.

Each concentration level and control were analysed via GC-MS method on day 0 and day 28 using mixed samples of all replicates, each. The recovery rates were in the ranges from 73% to 112% of the nominal concentrations of the test item on day 0. For the test item concentration 12.5 mg/kg DW, a recovery rate of 61 % was found. As this test item concentration is far below the NOEC as well as EC10 value, its lower recovery rate has no impact on the outcome of the study. On day 28, the recovery rates were in the ranges from 18% to 37%.The method was validated according to SANCO/3029/99 rev.4 (2000) prior to the start of exposure.

After 28 days of exposure in soil, neither evident earthworm mortalities (< 10%) nor evident pathological symptoms or changes in the behaviour of adult earthworms were observed in the control and all test item concentrations. There were no statistically significant differences in earthworm body weights in all test item concentrations compared to the control. After a further four weeks, the reproduction rate (average number of juveniles produced) was 127 in the control and ranged from 106 to 168 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction in all treatment rates.

All validity criteria recommended by the test guidelines were fulfilled.

 

Endpoint	[mg test item/kg SDW]
LOECmortality, biomass, reproduction	> 200
NOECmortality, biomass, reproduction	≥ 200
EC-values reproduction (95 % Confidence Interval)	EC10: 92.2 (49.5 - >200)

Description of key information

OECD Guideline 222, GLP, key study, validity 2:

EC10 reproduction (Eisenia fetida): 92.2 mg test item /kg SDW - 95% Confidence Interval ( 49.5 - > 200) 

LOEL mortality, biomass, reproduction (Eisenia fetida) : > 200 mg test item /kg SDW

NOEL mortality, biomass, reproduction (Eisenia fetida) : > or =  200 mg test item /kg SDW

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for soil macroorganisms:

92.2 mg/kg soil dw

Additional information

Effects of registered substance on mortality, biomass and the reproductive potential of the earthworm species Eisenia fetida (Annelida, Lumbricidae) were determined according to OECD 222 (2016).

The study was conducted under static conditions over 8 weeks with the test item concentrations 6.25 - 12.5 – 25 – 50 – 100 – 200 mg test item/kg SDW. Each application rate was mixed into artificial soil containing 5 % peat. A control including untreated artificial soil was tested under the same conditions as the test item treatments. 80 test organisms were inserted into 8 control replicates. 40 test organisms were divided into 4 replicates for each test item treatment (10 earthworms per replicate). They had an individual body weight between 0.40 and 0.54 g at experimental starting.

Each concentration level and control were analysed via GC-MS method on day 0 and day 28 using mixed samples of all replicates, each. The recovery rates were in the ranges from 73% to 112% of the nominal concentrations of the test item on day 0. For the test item concentration 12.5 mg/kg DW, a recovery rate of 61 % was found. As this test item concentration is far below the NOEC as well as EC10 value, its lower recovery rate has no impact on the outcome of the study. On day 28, the recovery rates were in the ranges from 18% to 37%.The method was validated according to SANCO/3029/99 rev.4 (2000) prior to the start of exposure.

After 28 days of exposure in soil, neither evident earthworm mortalities (< 10%) nor evident pathological symptoms or changes in the behaviour of adult earthworms were observed in the control and all test item concentrations. There were no statistically significant differences in earthworm body weights in all test item concentrations compared to the control. After a further four weeks, the reproduction rate (average number of juveniles produced) was 127 in the control and ranged from 106 to 168 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction in all treatment rates.

All validity criteria recommended by the test guidelines were fulfilled.

Under the test conditions, the NOEC was determined to be at least the nominal concentration 200 mg test item/kg SDW and EC10 at 92.2 mg test item/kg SDW