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Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-04-30 to 2021-09-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2016

Deviations:

yes

Remarks:

Food was provided on day 0 (test start) instead of day 1 due to good experience with this procedure. Soil moisture: see "Any other information on materials and methods incl. tables", pH-Values, Moisture Room Temperature: see Test conditions, Temperature

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test item LICOCARE RBW 300 FL TP
Batch number DEF2114527
Content (certified) 99.75% w/w
CAS No. Not definable
Appearance yellow solid flakes
Water solubility < 1g/L (OECD 105)
Stability under Not specified
test conditions
Expiry date 2022-11-05 (Minimum durability)
Recommended storage Room temperature, in tightly closed container in cool
and well ventilated area, keep away from a source of
ignition

Vehicle:

yes

Remarks:

Quartz sand

Details on preparation and application of test substrate:

The test item was treated with a mortar.
The respective test item amount for each test item concentration was weighed out and mixed with quartz sand (10 g per replicate).
The spiked quartz sand (40 g) was given to the artificial soil (2360 g sdw). Demineralised water (308 g) was added to the artificial soil to adjust the humidity of the artificial soil to a moisture of 54 % of the WHCmax.
Subsequently, the test medium was thoroughly mixed to ensure a homogenous distribution and about 600 g SDW were filled into each test vessel.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

Test system
Eisenia fetida (Annelida, Lumbricidae)

Reason for the selection
Eisenia fetida is suitable for this kind of study and is one of the recommended species according to the guideline.

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.

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 1 month.

Body weight (actual)
0.40 to 0.54 g


Adaption
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.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Test temperature:

Temperature 20 +- 2 °C

The room temperature temporarily deviated from the nominal room temperature for > 8 h in total about 216 hours which is approx. 16 % of the overall test duration (see "Any other information on materials and methods incl. tables",Environmental Conditions). However, its impact on the soil temperature is judged to be negligible as shown by the fulfilment of all validity criteria. 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 5.61 (measured on day -7 before the start of exposure).
For the pH-values at test start and end please refer to "Any other information on materials and methods incl. tables",pH-Values, Moisture and WHCmax of the Test Media.

Moisture:

Two days before test start the artificial soil (20000 g) was pre-moistened with 2334 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.

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.
For the moisture content of the test medium at test start and test end, please refer to "Any other information on materials and methods incl. tables", pH-Values, Moisture and WHCmax of the Test Media).

Details on test conditions:

Test duration
8 weeks

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

Test medium
Artificial soil consisted of the following components:
- 10 % peat, air-dried and finely ground
- 20 % kaolin, kaolinite content > 30 %
- 69 % air-dried quartz sand (sand with > 50 % particle size of
0.05 - 0.2 mm)
- 0.35 % 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 test start the artificial soil (20000 g) was pre-moistened with 2334 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 600 g SDW 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.

Temperature 20 +- 2 °C
Photoperiod 16 h light and 8 h dark
Light intensity 400 - 800 lx


Feeding
The earthworms were fed with air-dried cattle manure weekly during the first 4 weeks (see "Any other information on materials and methods incl. tables", Feeding and Additional Moistening During the Test).
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 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 (see "Any other information on materials and methods incl. tables", Feeding and Additional Moistening During the Test and pH-Values. Moisture and WHCmax of the Test Media).

Nominal and measured concentrations:

Five test item concentrations were tested:

62.5 - 125 - 250 - 500 - 1000 mg product/kg soil dry weight
(factor 2)
The application rates are determined based on the results of an
acute earthworm toxicity test with the same test item (Winkelmann, G., 2018, Study ID: 180706CM / RRA18315).

Reference substance (positive control):

yes

Remarks:

CARBENDAZIM

Key result

Duration:

57 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

57 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

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:

growth

Key result

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

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:

mortality

Details on results:

-No earthworm mortality was observed in the control and all test item concentrations.
-No pathological symptoms or changes in the behaviour of adult earthworms were observed in the control 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 biomass in all treatment rates compared to the control.
-The reproduction rate (average number of juveniles produced) was 155 in the control and ranged from 141 to 159 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction at all treatment rates.
The coefficient of variation calculated for the reproduction of the control was 11.0% and thus lower than 30 %.

Results with reference substance (positive control):

The test is conducted once within twelve months. The most recent test was carried out from 2021-02-19 to 2021-04-15 using artificial soil containing 10 % peat (Study ID 72102IRS / RBN70070).
According to OECD 222 significant effects of Carbendazim should be observed between 1 and 5 mg a.i./kg SDW.
For details please refer to "Any other information on results incl. tables",Data of Reference Item.

Reported statistics and error estimates:

Adult Mortality
The arithmetic mean and NOEC were determined.
Since no mortality occurred no further computations were performed.


Biomass
- Shapiro-Wilk’s Test on Normal Distribution
- Levene’s Test on Variance Homogeneity (with Residuals)
- Trend Analysis on Contrasts (Monotonicity of Concentration/Response)
- Dunnett’s Multiple Sequential t-test Procedure
The alpha-value (acceptable probability of incorrectly concluding that there is a difference) was alpha = 0.05. For the endpoint of biomass, the arithmetic mean and the variance (coefficient of variation) per treatment and control were calculated.


Reproduction
- Shapiro-Wilk’s Test on Normal Distribution
- Levene’s Test on Variance Homogeneity (with Residuals)
- Trend Analysis on Contrasts (Monotonicity of Concentration/Response)
- Dunnett’s Multiple t-test Procedure

The alpha-value (acceptable probability of incorrectly concluding that there is a difference) was alpha = 0.05. For the endpoint of reproduction, the arithmetic mean and the variance (coefficient of variation) per treatment and control were calculated. Since the slope of the relationship was found to be not significant, no EC and confidence limits are provided.

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

Application rate	Replicate
[mg test item/kg  SDW]	1	2	3	4	5	6	7	8	Mean
Control	0	0	0	0	0	0	0	0	0
62.5	0	0	0	0					0
125	0	0	0	0					0
250	0	0	0	0					0
500	0	0	0	0					0
1000	0	0	0	0					0

 

Pathological Symptoms and Changes in Behaviour

Application rate					Replicate
[mg test item/kg  SDW]	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
62.5	A	10/10	10/10	10/10	10/10
125	A	10/10	10/10	10/10	10/10
250	A	10/10	10/10	10/10	10/10
500	A	10/10	10/10	10/10	10/10
1000	A	10/10	10/10	10/10	10/10

A = no obvious pathological symptoms

 

Body Weight Changes of the Adult Earthworms

Application rate	Replicate	Mean body weights per replicate		Mean body weight change of earthworms
[mg test item/kg  SDW]		Test start	28 days	Per replicate		Mean ± SD		Sig.
		[g]	[g]	[g]	[%]	[g]	[%]
Control	1	0.47	0.47	0.00	0.0	0.03 ± 0.02	5.55 ± 5.02	–
	2	0.48	0.47	-0.01	-2.1
	3	0.47	0.51	0.04	8.5
	4	0.48	0.54	0.06	12.5
	5	0.48	0.49	0.01	2.1
	6	0.47	0.50	0.03	6.4
	7	0.47	0.51	0.04	8.5
	8	0.47	0.51	0.04	8.5
62.5	1	0.48	0.46	-0.02	-4.2	0.02 ± 0.03	4.25 ± 6.69	No
	2	0.48	0.49	0.01	2.1
	3	0.47	0.51	0.04	8.5
	4	0.47	0.52	0.05	10.6
125	1	0.47	0.50	0.03	6.4	0.04 ± 0.02	8.40 ± 3.72	No
	2	0.48	0.53	0.05	10.4
	3	0.48	0.54	0.06	12.5
	4	0.47	0.49	0.02	4.3
250	1	0.48	0.55	0.07	14.6	0.07 ± 0.02	14.2 ± 4.45	No
	2	0.47	0.54	0.07	14.9
	3	0.47	0.56	0.09	19.1
	4	0.48	0.52	0.04	8.3
500	1	0.47	0.52	0.05	10.6	0.04 ± 0.02	7.38 ± 5.03	No
	2	0.48	0.48	0.00	0.0
	3	0.48	0.52	0.04	8.3
	4	0.47	0.52	0.05	10.6
1000	1	0.47	0.53	0.06	12.8	0.03 ± 0.02	6.90 ± 4.07	No
	2	0.48	0.50	0.02	4.2
	3	0.48	0.50	0.02	4.2
	4	0.47	0.50	0.03	6.4

SD = Standard deviation
Sig. = Statistical Significance (Dunnett’s Multiple Sequential t-test Procedure, alpha = 0.05) compared to control

 

 

Reproduction Rate (Number of Juveniles after 8 Weeks)

Application rate	Replicate	Number of juveniles	Mean ± SD	CV               [%]	[%]	Sig.
[mg test item/kg SDW]					of control
Control	1	182	155 ± 17.1	11.0	-	-
	2	181
	3	145
	4	140
	5	152
	6	147
	7	141
	8	148
62.5	1	135	150 ± 27.9	18.6	96.8	No
	2	132
	3	191
	4	140
125	1	191	159 ± 26.1	16.4	103	No
	2	135
	3	141
	4	170
250	1	135	141 ± 17.7	12.5	91.0	No
	2	165
	3	142
	4	123
500	1	137	154 ± 14.8	9.58	99.4	No
	2	152
	3	173
	4	154*
1000	1	134	150 ± 23.1	15.4	96.8	No
	2	134
	3	183
	4	149

SD = Standard deviation CV = Coefficient of variation
Sig. = Statistical Significance (Dunnett’s Multiple t-test Procedure, α = 0.05) compared to the control
*) One adult worm was found on day 56, not taken into consideration

 

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, neither earthworm mortalities nor 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 biomass in all test item concentrations compared to the control. After a further four weeks, the reproduction rate (average number of juveniles produced) was 155 in the control and ranged from 141 to 159 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction in all treatment rates compared to the control. No effect values could be determined for biomass, reproduction and mortality, as no effects occurred up to 1000 mg test item/kg SDW.
All validity criteria recommended by the test guidelines were fulfilled.

Executive summary:

Effects of LICOCARE RBW 300 FL TP (batch number: DEF2114527) on mortality, biomass and the reproductive potential of the earthworm species Eisenia fetida (Annelida, Lumbricidae) were determined according to OECD 222 (2016) from 2021-04-30 to 2021-09-10 with the definitive exposure phase from 2021-07-15 to 2021-09-09 at the test facility in 31157 Sarstedt, Germany.

The study was conducted under static conditions over 8 weeks with the test item concentrations 62.5 – 125 – 250 – 500 – 1000 mg LICOCARE RBW 300 FL TP/kg SDW. Each application rate was mixed with an appropriate amount of quartz sand followed by mixing into artificial soil containing 10 % 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.

After 28 days of exposure in soil, neither earthworm mortalities nor 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 biomass in all test item concentrations compared to the control. After a further four weeks, the reproduction rate (average number of juveniles produced) was 155 in the control and ranged from 141 to 159 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction in all treatment rates compared to the control. No effect values could be determined for biomass, reproduction and mortality, as no effects occurred up to 1000 mg test item/kg SDW.

All validity criteria recommended by the test guidelines were fulfilled.

 

Summary of All Observed Effects in the Application rates

Effects	LICOCARE RBW 300 FL TP
Application rates	Control	62.5	125	250	500	1000
[mg product/kg  SDW]
Mean Mortality of adult earthworms [%]	0	0	0	0	0	0
Biomass [mean body-weight change* after 28 days of exposure in %]	5.6	4.3	8.4	14	7.4	6.9
Reproduction	-	96.8	103	91.0	99.4	96.8
[% of control]
Summary of all effects	-	No	No	No	No	No
[Statistical significance compared to the control]

*) increase of body weight

 

 

NOEC, LOEC and EC-values

Endpoint	[mg test item/kg  SDW]
LOECmortality, biomass, reproduction	> 1000
NOECmortality, biomass, reproduction	≥ 1000

 

Reference 2

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-11-14 to 2018-11-29 with the definitive exposure phase from 2018-11-14 to 2018-11-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Version / remarks:

1984

Deviations:

yes

Remarks:

6 of 12 in total used glass vessels had the wrong volume (2 L instead of 1.5 L). This deviation is considered to have no impact on quality and integrity of the study.

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

yes

Remarks:

Quartz sand

Details on preparation and application of test substrate:

The test item was treated with a mortar.
The respective test item amount was weighed out (nominal weight: 3330 mg; actual weight: 3330.3 mg) for the limit test item concentration and mixed with quartz sand (10 g per replicate). The spiked quartz sand was given to the artificial soil. Demineralised water was added to the artificial soil to adjust the humidity of the artificial soil to a moisture of 54 % of the maximum water holding capacity. Subsequently, the test medium was thoroughly mixed to ensure a homogenous distribution and about 555 g SDW were filled into each test vessel.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Eisenia fetida (Annelida, Lumbricidae)
- Source: Breeding stock culture maintained at the test facility
- Age at test initiation (mean and range, SD): Adult
- Weight at test initiation (mean and range, SD): 0.41 +/- 0.03

ACCLIMATION
- Acclimation period: 2 days
- Acclimation conditions (same as test or not): same as test
- Health during acclimation (any mortality observed): no mortality observed

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

yes

Total exposure duration:

14 d

Test temperature:

17.5 - 22 °C

pH:

Test item concentration
[mg/kg SDW]
pH-value

day 0 day 14
Control 5.99 5.64
1000 5.97 5.60

Moisture:

Test item concentration
[mg/kg SDW]

Moisture [%] of DW
day 0 day 14
Control 26.0 24.9
1000 26.1 24.8

Details on test conditions:

TEST SYSTEM
- Test containers Glass vessels (volume 1.5 L and 2 L, used for preserving) were used. During the test, transparent and perforated lids enabled sufficient gas exchange and light input and prevented drying of the test substrate.
- Amount of soil or substrate: 555 g soil dry weight
- No. of organisms per container (treatment): 10 per replicate
- No. of replicates per treatment group: 6
- No. of replicates per control: 6

Artificial soil:
- Artificial soil consisted of the following components:
- 10 % Peat, air-dried and finely ground
- 20 % Kaolin, kaolinite content > 30 %
- 69 % Air-dried quartz sand (sand with > 50 % particles size
of 0.05 to 0.2 mm)
- 0.38 % calcium carbonate (CaCO3) to adjust the pH to 6.0 ±
0.5
The soil moisture content and the WHCmax were determined prior to experimental starting.
Two days before earthworm insertion into the test vessels the artificial soil (7840 g sdw) was pre-moistened with 883 g deionised water to achieve a moisture of 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.



OTHER TEST CONDITIONS
- Photoperiod: 16 h
- Light intensity: 529 +/- 84

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The live weight of the earthworms was determined individually on the day of application (day 0) and on day 14.
The mean weight change was assessed on day 14 after application.
Mortality, behaviour and morphological changes of the earthworms were recorded 7 and 14 days after application.


VEHICLE CONTROL PERFORMED: no

TEST CONCENTRATIONS
- Spacing factor for test concentrations: none, limit test
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations: 1 - 10 - 100 - 1000 mg/kg soil dry weight
- Results used to determine the conditions for the definitive study:
Range Finding Test (14 days): Mortality and Change of Live Weight
Concentration Mortality of adult earthworms Change of Live weight
[mg test item/kg SDW] [%] [%]
Control 0 -17
1 0 -15
10 0 -19
100 0 -14
1000 0 -13

Nominal and measured concentrations:

1000 mg/kg soil dry weight

Reference substance (positive control):

yes

Remarks:

2-Chloroacetamide

Duration:

14 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Mortality, behaviour, pathological symptoms, change of body weight

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Mortality, behaviour, pathological symptoms, change of body weight

Details on results:

- Mortality at end of exposure period: No mortality was observed in the control and the limit test item concentration 1000 mg/kg SDW after 14 days of exposure
- Total mass of adults at beginning of test: Control: 24.7 g; 1000 mg/kg soil dry weight: 24.8 g
- Changes in body weight of live adults (% of initial weight) at end of exposure period: In terms of body weight loss, no statistically significant effect was observed in the limit test item concentration after 14 days of exposure compared to the control.
- Morphological abnormalities: no pathological symptoms or changes in earthworm behaviour were observed in the control and the limit test item concentration after 14 days of exposure

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels:
NOEC and LC-Value after 14 d for the Reference Item 2-Chloroacetamide
LC-Value /
NOEC Concentration
[mg reference item/kg SDW] 95 % - Confidence Interval
[mg reference item/kg SDW]
NOEC 30 –
LC50 42.3 n.d.*
*) not determined due to mathematical reasons (3 reference item concentrations)

Reported statistics and error estimates:

NOEC, LOEC and statistical Since no mortality was observed, no further computation was
analyses carried out.
The NOEC/LOEC for biomass was determined using a Two-sample t-test procedure for determination of statistically significant differences in biomass compared to the control. When running the test, a Shapiro-Wilk’s Test on Normal Distribution and Brown-Forsythe test on Equal Variance were done first. The alpha-value (acceptable probability of incorrectly concluding that there is a difference) is alpha = 0.05.

Earthworm Mortality in [%] after 7 and 14 Days of Exposure

					Mortality [%]
Test item concentration [mg/kg SDW]	Day 7							Day 14
	1	2	3	4	5	6	MV	1	2	3	4	5	6	MV
Control	0	0	0	0	0	0	0	0	0	0	0	0	0	0
1000	0	0	0	0	0	0	0	0	0	0	0	0	0	0

MV = mean value

Earthworm Behaviour and Pathological Symptoms after 7 and 14 Days of Exposure

Test item concentration [mg/kg SDW]	Effect	Replicate
		1	2	3	4	5	6
		Day 7
Control	A)	10/10	10/10	10/10	10/10	10/10	10/10
1000	A)	10/10	10/10	10/10	10/10	10/10	10/10
		Day 14
Control	A)	10/10	10/10	10/10	10/10	10/10	10/10
1000	A)	10/10	10/10	10/10	10/10	10/10	10/10

Mean Live Weight and Mean Loss of Body Weight

	Mean live weight
Test item  concentration  [mg/kg SDW]	Test start MV±SD [g/worm]	Test end MV±SD [g/worm]	Inhibition [%]*	Statistically significant vs.  Control
Control	0.41 ± 0.03	0.43 ± 0.05	-5	–
1000	0.41 ± 0.03	0.42 ± 0.04	-2	No

MV = Mean value           SD = Standard deviation     

Validity criteria fulfilled:

yes

Conclusions:

No mortality and no pathological symptoms or changes in earthworm behaviour were observed in the control and the limit test item concentration after 14 days of exposure. Therefore, the NOEC was determined to be 1000 mg test item/kg SDW.
In terms of body weight loss, no statistically significant effect was observed in the limit test item concentration after 14 days of exposure compared to the control. Consequently, the NOEC was determined to be 1000 mg test item/kg SDW.

Executive summary:

The acute effects of Licocare RBW 300 on the earthworm Eisenia fetida were determined according to the OECD Guideline 207 at the test facility in 31157 Sarstedt, Germany from 2018-11-14 to 2018-11-29 with thedefinitive exposure phase from 2018-11-14 to 2018-11-28. The study was conducted under static conditions for 14 days with a limit test item concentration of 1000 mg/kg SDW mixed into artificial soil containing 10 % peat. Artificial soil without test item was used as control. 60 test organisms with an individual weight between 0.34 - 0.49 g, divided into six replicates, were tested per limit test item concentration and control.

No mortality and no pathological symptoms or changes in earthworm behaviour were observed in the control and thelimit test item concentration after 14 days ofexposure. Therefore, the NOEC was determined to be 1000 mg test item/kg SDW .

In terms of body weight loss, no statistically significant effect was observed in the limit test item concentration after 14 days of exposure compared to the control. Consequently, the NOEC was determined to be 1000 mg test item/kg SDW .

 

All validity criteria of the test guideline were fulfilled.

 

Summary of all Effects

Effects	Licocare RBW 300 [mg/kg SDW]
LOEC Mortality, behaviour, pathological symptoms,               change of body weight	> 1000
NOEC Mortality, behaviour, pathological symptoms,             change of body weight	1000

Description of key information

OECD 207

The acute effects of Licocare RBW 300 on the earthwormEisenia fetidawere determined according to the OECD Guideline 207

No mortality and no pathological symptoms or changes in earthworm behaviour were observed in the control and the limit test item concentration after 14 days of exposure. Therefore, the NOEC was determined to be 1000 mg test item/kg SDW.

In terms of body weight loss, no statistically significant effect was observed in the limit test item concentration after 14 days of exposure compared to the control. Consequently, the NOEC was determined to be 1000 mg test item/kg SDW.

 

OECD 222

Under the conditions of this study, neither earthworm mortalities nor 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 biomass in all test item concentrations compared to the control. After a further four weeks, the reproduction rate (average number of juveniles produced) was 155 in the control and ranged from 141 to 159 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction in all treatment rates compared to the control. No effect values could be determined for biomass, reproduction and mortality, as no effects occurred up to 1000 mg test item/kg SDW.
All validity criteria recommended by the test guidelines were fulfilled.

Key value for chemical safety assessment

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

1 000 mg/kg soil dw