Registration Dossier

Diss Factsheets

Ecotoxicological information

Toxicity to soil macroorganisms except arthropods

Currently viewing:

Administrative data

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:
17 April 2013 to 15 November 2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
Version / remarks:
2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
ISO 11268-1 (Effects of Pollutants on Earthworms. 1. Determination of Acute Toxicity Using Artificial Soil Substrate)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
yes
Remarks:
Acetone
Details on preparation and application of test substrate:
- Method of mixing into soil: Since the test material is poorly soluble in water, it was dissolved in acetone and 2 mL of the test material solution were applied on 42 g quartz sand for each test material group (corresponding to 10 g quartz sand per replicate. The sand containing the test material was mixed into the pre-moistened test substrate. Thorough mixing was conducted at high speed with an electric drill. Afterwards 104 g deionised water (208 g for the control group) were added to the test substrate and thoroughly mixed. Immediately after mixing, 740 g of the test substrate (corresponding to 590 g dry substrate plus 10 g quartz sand) were placed into each of the test vessels.
- Method of application to soil surface: The test material was applied to the substrate by mixing.
- Controls: For the control group, 4 mL of acetone were pipetted on 84 g quartz sand and then applied to the pre-moistened substrate.
- Chemical name of vehicle: Acetone
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM:
- Common name: Earthworm
- Source: From a healthy laboratory rearing stock maintained in the testing facility.
- Age at test initiation: They were at least two months old, but not older than 1 year with a clitellum. The age of the worms used for the test did not differ by more than four weeks.
- Weight at test initiation: The individual weights of the earthworms were recorded after washing and drying them shortly before use. The mean weight at the start of the test ranged from 481.1 to 487.6 mg/worm, which is within the selection criteria for test organisms of 316 and 600 mg.
- Culture: The earthworms were cultured in a climatic chamber at 20 °C ± 2 °C, under long day conditions (16 h light/8 h darkness) with a light intensity between 400 and 800 Lux. They were kept in covered plastic boxes containing approx. 200 - 300 individuals in potting soil or peat mixed with non-contaminated horse manure. Synchronisation was reached by selecting the adult earthworms every four weeks from the soil and transferring them to new containers.

ACCLIMATION
- Acclimation period: Two days before the test started, the adult earthworms were selected and transferred from the rearing medium into moist, untreated artificial soil for acclimatisation.
- Health during acclimation: The test organisms were healthy, indicated by dark brown colour and high activity when put under light or in warm water.

Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
56 d
Remarks:
Mortality and effects on bodyweight were determined on day 28 after application. The reproductive output was calculated by taking into account the number of offspring hatched from the cocoons until day 56 after application.
Test temperature:
19.2 - 20.3 °C
pH:
pH 5.5 at test initiation, and pH 6.1 - 6. 3 at test termination.
Moisture:
Water content at test initiation 25.0 % - 25.4 % and 25.0 % - 27.8 % at test termination.
Details on test conditions:
TEST SYSTEM
- Test container: The test containers were closed with a perforated lid to allow gaseous exchange between the medium and the atmosphere.
- Amount of soil or substrate: 740 g of the test substrate (corresponding to 590 g dry substrate plus 10 g quartz sand) were placed into each of the test vessels.
- No. of organisms per container: 10 randomly selected earthworms meeting the test criteria.
- No. of replicates per treatment group: 4 replicates/test material group, each with 10 earthworms
- No. of replicates per control: 8 replicates/test material group, each with 10 earthworms
- Control: Acetone-treated quartz sand

SOURCE AND PROPERTIES OF SUBSTRATE
- Artificial Soil Components:
• 10 % sphagnum peat (close to pH 5.5 - 6.0; no visible plant remains, finely ground, air dry);
• 20 % kaolin clay (kaolinite content above 30 %);
• Approx. 70 % industrial sand (fine sand dominant with more than 50 % of the particles between 50 and 200 microns);
• Approx. 0.15 % calcium carbonate - precipitated extra pure (the soil pH is adjusted to 6.0 ± 0.5 at the start of the test before the addition of the test material).
- Pretreatment of soil: The dry components were blended and mixed thoroughly in an electric mixer for almost a day. After mixing, the water holding capacity was determined for each batch of artificial soil and was given as mean value.
After mixing the components, i.e. before pre-moistening, 10 g air-dried finely ground cow manure per 1 kg substrate were added.
The water holding capacity of the substrate used for the test was determined to be 40.68 %. In order to achieve a soil water content of approximately 55 % of the soil water holding capacity, approx. 134.2 g of deionised water were added per 600 g dry artificial soil, i.e. per replicate. 10 g dried and finely ground cow manure were added per kg dry artificial soil, corresponding to one percent of the dry mass. For pre-moistening, the total amount of 6.735 kg deionised water was added to 36.260 kg dry soil one day before application. The pre-moistened soil was stored in a closed container until use. The amount of 740 g of quartz sand which was needed for application complemented to the total amount of 37.0 kg dry substrate. On the day of application, 5.877 kg pre-moistened substrate (corresponding to 4.916 kg dry substrate) were weighed in for the control group whereas 2.938 kg pre-moistened substrate (corresponding to 2.458 kg dry substrate) were weighed in per test material group. Application of 84 g (control group) and 42 g (test material groups) of quartz sand complemented to a total amount of 5.000 kg (control group) and 2.500 kg (test material groups) dry substrate, respectively. 208 g deionised water (control group) or 104 g deionised water (test material groups) were added for final moistening.
- Soil Water Content Determination: At the beginning of the test and 56 days after application approx. 10 g equivalents from each treatment group were removed to determine soil water content. At the end of the test the water content should not differ more than 10 % from that at the beginning of the test. Soil water content was determined by weighing samples before and after drying overnight at 105 °C, following DIN ISO 11465 (1996).
- Soil pH Determination: Soil pH (in CaCl2), determined with a calibrated electrode following DIN ISO 10390 (1997), was recorded for each treatment group from samples taken on the day of application and 56 days after application.
- Maximum water holding capacity: The water holding capacity was determined as follows: Three crucibles with an unglazed lower section were incubated for 30 minutes in a water bath for saturation. Afterwards the crucibles were weighed and 50 g of dry artificial soil was added. After at least 12 h incubation in a water bath, the crucibles were placed on filter paper for drainage of free water for 2 h. Finally, the overall weight of the crucibles and the artificial soil was recorded.
The water holding capacity was calculated using the formula:

WC = mCA – mCB

WHCmax = ((WC x 100 %) / (IW x DW)) x 100

WC = Amount of water in the soil [g]
mcA = Weight of the crucible + soil after saturation [g]
mCB = Weight of the crucible + soil before saturation [g]
WHCmax = Maximum water holding capacity [%]
IW = Initial soil weight [g]
DW = Soil dry weight of air dried soil [%]

OTHER TEST CONDITIONS
- Photoperiod: 16 h light/ 8 h darkness
- Light intensity:400 - 800 lux.
- Feeding and watering procedure: One day after application, the earthworms were fed by uniformly distributing 4 g finely ground cow manure per vessel on the soil surface. The manure powder was then moistened with 4 g deionised water sprayed on the soil surface. On day +7, +14 and +21 after test start, food was strewn onto the soil surface depending on the feeding activity. On the 28-day assessment, after adult worms were removed, 4 g food was added for the reproduction test and the test units were moistened with 4 g deionised water per vessel. Offspring was not fed further during the remaining 28 days of the study. The moisture of the substrate was adjusted weekly by reweighing the test units taking into consideration the respective amount of added food.
- Worms were placed onto the surface of the test substrate in each container in a non-systematic manner.

EFFECT PARAMETERS MEASURED:
- Mortality, behavioural abnormalities and pathological symptoms were recorded 28 days after application. The assessment started with the control treatment, followed by increasing concentrations of the test material.
For the examination, each test unit was emptied separately on a stainless steel tray. The earthworms were sorted out of the medium and their reaction to a gentle mechanical stimulus at the anterior end was tested. Individuals with no reaction were assumed to be dead. Missing earthworms were assumed to have degraded by autolysis and therefore were counted as dead. Adults were removed and the total weight of the surviving earthworms per replicate was recorded. After the assessment the artificial soil, containing all cocoons that had been produced, was returned to the test containers.
- Reproduction Test: To allow the offspring to develop from the cocoons the test containers were incubated for an additional period of 28 days under test conditions. The feeding was continued as described below. After 56 days the test was terminated and the number of offspring was determined.
The extraction of juvenile worms from the soil substrate was performed by placing the test containers in a water bath at approximately 55 °C. After a period of approximately 20 min, the juvenile worms appearing on the substrate surface were collected and counted.
- Reported Endpoints: Endpoints reported are mortality, body weight change and reproduction given as NOEC (no observed effect concentration) and LOEC (lowest observed effect concentration) as well as the EC10, EC20, and EC50 for reproduction. Additionally, changes in behaviour and morphology of the adult earthworms were recorded if observed. Mortality is given in percent per treatment group after four weeks. Reproduction was evaluated as number of juveniles per replicate and mean number per treatment group after eight weeks. Earthworm body weight was recorded individually at test initiation. After four weeks of exposure the earthworm body weight per replicate was recorded. Body weight change is reported as absolute weight change and in percent per treatment group.

VEHICLE CONTROL PERFORMED: Yes.
Nominal and measured concentrations:
3.3, 6.0, 10.8, 19.4 and 35.0 mg test material/ kg soil dry weight.
Reference substance (positive control):
yes
Remarks:
Carbendazim (formulation 'Twist WP 60 % w/w')
Key result
Duration:
56 d
Dose descriptor:
EC10
Effect conc.:
9 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: Confidence limits of 5.1 - 12.8 mg/kg soil dw.
Key result
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
10.8 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
56 d
Dose descriptor:
LOEC
Effect conc.:
19.4 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
56 d
Dose descriptor:
other: EC20
Effect conc.:
26.4 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: Confidence limits of 18.2 - 53.4 mg/kg soil dw.
Details on results:
- Mortality at end of exposure period: After 28 days of exposure, no mortality was observed in the control group and in the test material groups of 3.3, 10.8, 19.4 and 35.0 mg test material/ kg soil dry weight. At the test material concentration of 6.0 mg/kg soil dry weight one earthworm was missing and was therefore counted as dead, resulting in a mean mortality of 2.5 %.

- Changes in body weight of live adults (% of initial weight) at end of exposure period: The earthworm body weight was determined as the mean body weight per surviving worm. The body weight change was determined using the means of absolute body weight at start and after 28 days per replicate. Based on these mean values the body weight change per treatment was calculated.
After 28 days of exposure to the test material, the average body weight change of the adult earthworms was +23.2, +27.5, +19.9, +17.8 and +24.7 % in the test material groups of 3.3, 6.0, 10.8, 19.4 and 35.0 mg/kg soil dry weight. In the control group, the average body weight change was +27.3 % in relation to the initial weight.

- Reproduction capacity: The mean number of juveniles per replicate in the test material groups was 282.8, 298.3, 283.0, 233.3 and 242.5 for concentrations of 3.3, 6.0, 10.8, 19.4 and 35.0 mg/kg soil dry weight compared to 306.8 in the control group.
After homoscedasticity and normality had been assured, Williams test (descending order, α = 0.05) was conducted. This test revealed statistically significant decrease in the number of juveniles at 19.4 and 35.0 mg/kg soil dry weight.
Based on the results of the study, the NOEC and LOEC for reproduction was therefore determined as 10.8 and 19.4 mg/kg soil dry weight, respectively.
The EC10 was calculated as 9.0 mg/kg soil dry weight (lower confidence limit 5.1 mg/kg soil dry weight, upper confidence limit 12.8 mg/kg soil dry weight).
The EC20 was calculated as 26.4 mg/kg soil dry weight (lower confidence limit 18.2 mg/kg soil dry weight, upper confidence limit 52.4 mg/kg soil dryweight).
The EC50 for reproduction could not be calculated as none of the test material groups reached the threshold value of 50 % difference to the control.

- Behavioural abnormalities: No effects of the test material on earthworm behaviour could be detected in this study.
Results with reference substance (positive control):
The treatment with the reference material 'Twist WP 60 % w/w' (8.58 mg/kg soil dry weight, equivalent to 5.00 mg carbendazim/kg soil dry weight) tested as a separate study resulted in a statistically significant reduction in reproduction of E. fetida ssp. andrei of 96.3 % (Mann-Whitney test, p ≤ 0.05). There was no significant effect on mortality.

After 28 days no mortality of the adult earthworms was observed in the control group. In the test material group of 8.58 mg product/kg soil dry weight (equivalent to 5.00 mg a.i./kg soil dry weight), one earthworm was missing: resulting in a mean mortality of 1.25 %.
After 28 days of exposure to the test material of 8.58 mg product/kg soil dry weight (equivalent to 5.00 mg a.i./kg soil dry weight), the mean weight change of the adult worms was + 18.8 % in relation to the initial weight. In the control group, the mean body weight change was +27.8 % in relation to the initial weight.
The mean number of juveniles in the test material group of 8.58 mg product/kg soil dry weight (equivalent to 5.00 mg a.i./kg soil dry weight) was 4.6 compared to 123.8 juveniles in the control group. The number of juveniles was statistically significantly different from the control in the test material group of 8.58 mg product/kg soil dry weight (equivalent to 5.00 mg a.i./kg soil dry weight; Mann-Whitney Test, Exact, left-sided. p ≤ 0.05).
During the hand-sorting of the replicates for the validation of the extraction method revealed one juvenile in one replicate resulting in a efficiency of 99.9 %.
In conclusion the treatment with Twist WP 60 % w/w at a concentration of 8.58 mg product/kg soil dry weight (equivalent 10 5.00 mg a.i./kg soil dry weight) resulted in a statistically significant reduction in reproduction of Eisenia andrei (Mann-Whitney Test, Exact, left-sided, p ≤ 0.05). The validation of the extraction method resulted in 99.9 % efficiency and deemed to be suitable for juvenile extraction.
Reported statistics and error estimates:
The body weight change is reported as mean percent body weight change and as absolute weight change of earthworms 28 days after the treatment compared to the initial weight. The mean body weight change per treatment was determined using the means of worm weight per replicate. Reproduction is reported as number of juveniles per replicate. The data were tested for normality using Shapiro-Wilks test and for homoscedasticity using Bartlett's test. As the requirements of normality and homoscedasticity were met, Williams test (descending order, α = 0.05) was used to analyse the reproductive output data.
The EC50 for reproduction could not be calculated as none of the test material groups reached the threshold value of 50 % difference to the control. The EC10 and EC20 values for reproduction were calculated using Probit procedure, model: Normal.
Statistical calculations were done using SAS Version 9 .3 (2002 - 2010).

Summary of Effects of the test Material on Eisenia fetida ssp. Andreii Mortality, Body Weight Change and Reproduction.

Parameter

Concentration of the Test Material

[mg/kg Soil Dry Weight]

0 (Control)

3.3

6.0

10.8

19.4

35.0

Mortality [%]

0.0

0.0

2.5

0.0

0.0

0.0

Mean weight change [%]

27.3

23.2

27.5

19.9

17.8

24.7

Juveniles/replicate [mean]

306.8

282.8

298.3

283.0

133.3*

242.5*

Reproduction [%]

(deviation from the control)

-

-7.8

-2.8

-7.8

-24.0

-21.0

Endpoints [mg/kg soil dry weight]

NOECreproduction

10.8

LOECreproduction

19.4

EC10reproduction

9.0 (CL: 5.1 – 12.8)

EC20reproduction

26.4 (CL: 18.2 – 52.4)

* Statistically significantly different from the control (Williams test, descending order, p ≤ 0.05)

 

Mortality of Eisenia fetida ssp. Andrei after 28 Days of Exposure to the Test Substrate Treated

 

Treatment Group

 Concentration

[mg/kgsdw*]

Total number of Adult Earthworms Introduced

Total Number of DeadINot Recovered Adult Earthworms

Mean Mortality

[%]

Control (n=8)

0

80

0

0.0

 

 

 Test material (n=4)

3.3

40

0

0.0

6.0

40

1

2.5

10.8

40

0

0.0

19.4

40

0

0.0

35.0

40

0

0.0

 *sdw: Soil dry weight

Average Body Weight of Adult Earthworms After 28 Days of Exposure to the Test Substrate

 

Treatment Group

Concentration

[mg/kgsdw*]

At Test Start

After 28 Days

Mean Weight

[mg/worm]

 

±SD**

Mean Weight

[mg/worm]

 

±SD**

Mean Weight Change

[mg/worm]

Mean Weight Change

[%]

Control (n=8)

0

481.1

34.6

612.5

59.5

131.4

27.3

Test Material (n=4)

3.3

485.5

20.8

597.9

59.6

112.4

23.2

6.0

486.1

17.0

619.7

47.1

133.6

27.5

10.8

487.6

17.0

584.8

28.4

97.2

19.9

19.4

485.3

21.6

571.8

37.7

86.5

17.8

35.0

486.2

18.3

606.l

59.6

119.9

24.7

*sdw: Soil dry weight

** SD: Standard deviation

 

Mean Number of Juvenile Worms After 56 Days of Exposure to the Test Substrate

Treatment Group

Concentration

[mg/kg sdw]

Mean Number of Juveniles Per Replicate

± SD

CV

[%]

Reduction in Reproduction†

[%]

Water control

(n = 8)

0

306.8

39.3

12.8

-

Test material

(n = 4)

3.30

282.8

47.2

16.7

7.8

6.00

298.3

15.2

5.1

2.8

10.8

283.0

55.3

19.5

7.8

19.4

233.3*

34.8

14.9

24.0

35.0

242.5*

20.6

8.5

21.0

† Compared to the control.

* Statistically significantly different from the control (Williams test, descending order p 0.05).

CV: Coefficient of variation.

SD: Standard deviation.

sdw: Soil dry weight.

Detailed Number of Juvenile Worms after 56 Days of Exposure to the Test Substrate

Treatment Group

Concentration

[mg a.i./kg sdw]

Number of Juvenile Worms Per Replicate*

1

2

3

4

5

6

7

8

Control (n = 8)

0

303

326

314

297

243

351

356

264

Test material

(n = 4)

3.3

252

327

319

233

-

-

-

-

6.0

319

283

298

293

-

-

-

-

10.8

332

329

227

244

-

-

-

-

19.4

191

219

266

257

-

-

-

-

35.0

213

261

249

247

-

-

-

-

*10 adult worms were introduced per replicate.

sdw = soil dry weight.

Validity Criteria

All required validity criteria were met.

Study Validity Check for Parameters in the Control Group

Parameter

Required

Observed

Mortality

10 %

0 %

Number of juveniles per vessel

30

243 - 356

Coefficient of variance (cv) of reproduction

30 %

12.8 %

 

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study, there was no effect on mortality or on parental reproduction after 56 days of exposure to the test material at up to 35 mg/kg soil.
The NOEC and LOEC for reproduction of Eisenia fetida ssp. andrei was determined as 10.8 and 19.4 mg/kg soil dry weight, respectively.
The EC10 and EC20 were calculated as 9.0 mg/kg soil dry weight and 26.4 mg test material/kg soil dry weight, respectively.
The EC50 for reproduction exceeded the top dose level of 35 mg test material/kg soil dry weight and can therefore be expressed as > 35 mg test material/kg soil dry weight.
Executive summary:

The objective of the study was the assessment of the side effects of the test material on reproduction and growth of Eisenia fetida ssp. andrei and the determination of the NOEC (no observed effect concentration), the LOEC (lowest observed effect concentration) and the determination of the EC10, EC20 and EC50, where possible. The test was performed in an artificial soil under laboratory conditions according to OECD 222 (2004), ISO Guideline 11268-2 (1998).

Mortality and effects on body weight were determined on day 28 after application. The reproductive output was calculated by taking into account the number of offspring hatched from the cocoons until day 56 after application.

A toxic reference material (carbendazim) was tested in the testing facility in a separate study to confirm the sensitivity under the test conditions of the test organisms against compounds with known effects. The test material was applied to the substrate by mixing. For each treatment group four test vessels were prepared (control: eight test vessels) and ten adult earthworms (not older than one year with clitellum) per test vessel were added.

Aliquots treated with acetone served as control.

Under the conditions of the study, there was no effect on mortality or on parental reproduction after 56 days of exposure to the test material at up to 35 mg/kg soil.

The NOEC and LOEC for reproduction of Eisenia fetida ssp. andrei was determined as 10.8 and 19.4 mg/kg soil dry weight, respectively.

The EC10 and EC20 were calculated as 9.0 mg/kg soil dry weight and 26.4 mg test material/kg soil dry weight, respectively. The EC50 for reproduction exceeded the top dose level of 35 mg test material/kg soil dry weight and can therefore be expressed as > 35 mg test material/kg soil dry weight.

Description of key information

Stojanowitsch (2014)

Under the conditions of the study, there was no effect on mortality or on parental reproduction after 56 days of exposure to the test material at up to 35 mg/kg soil.

The NOEC and LOEC for reproduction of Eisenia fetida ssp. andrei was determined as 10.8 and 19.4 mg/kg soil dry weight, respectively.

The EC10 and EC20 were calculated as 9.0 mg/kg soil dry weight and 26.4 mg test material/kg soil dry weight, respectively.

The EC50 for reproduction exceeded the top dose level of 35 mg test material/kg soil dry weight and can therefore be expressed as > 35 mg test material/kg soil dry weight.

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for soil macroorganisms:
10.8 mg/kg soil dw

Additional information

Stojanowitsch (2014)

The objective of the study was the assessment of the side effects of the test material on reproduction and growth of Eisenia fetida ssp. andrei and the determination of the NOEC (no observed effect concentration), the LOEC (lowest observed effect concentration) and the determination of the EC10, EC20 and EC50, where possible. The test was performed in an artificial soil under laboratory conditions according to OECD 222 (2004), ISO Guideline 11268-2 (1998). The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

Mortality and effects on body weight were determined on day 28 after application. The reproductive output was calculated by taking into account the number of offspring hatched from the cocoons until day 56 after application.

A toxic reference material (carbendazim) was tested in the testing facility in a separate study to confirm the sensitivity under the test conditions of the test organisms against compounds with known effects. The test material was applied to the substrate by mixing. For each treatment group four test vessels were prepared (control: eight test vessels) and ten adult earthworms (not older than one year with clitellum) per test vessel were added.

Aliquots treated with acetone served as control.

Under the conditions of the study, there was no effect on mortality or on parental reproduction after 56 days of exposure to the test material at up to 35 mg/kg soil.

The NOEC and LOEC for reproduction of Eisenia fetida ssp. andrei was determined as 10.8 and 19.4 mg/kg soil dry weight, respectively.

The EC10 and EC20 were calculated as 9.0 mg/kg soil dry weight and 26.4 mg test material/kg soil dry weight, respectively. The EC50 for reproduction exceeded the top dose level of 35 mg test material/kg soil dry weight and can therefore be expressed as > 35 mg test material/kg soil dry weight.