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Toxicity to soil macroorganisms except arthropods

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Reference
Endpoint:
toxicity to soil macroorganisms except arthropods: short-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-10-04 to 2018-10-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 207 (Earthworm, Acute Toxicity Tests)
Version / remarks:
1984
Deviations:
yes
Remarks:
The temperature was slightly above the required range for about 3 hours.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on preparation and application of test substrate:
The respective test item amount was weighed out for a stock solution (nominal: 4440 mg/200 mL, actual: 4440.3 mg/200 mL) and dispersed in demineralised water. The stock solution was further diluted to the respective application solutions. The application solution and additional demineralised water were 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 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): 0.46 +/- 0.04 g

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:
no
Total exposure duration:
14 d
Test temperature:
19 - 23 °C
The temperature was slightly above the required range for about 3 hours.
pH:
Test item concentration pH-value
[mg/kg SDW] day 0 day 14
Control 5.96 5.89
3.16 5.99 5.82
10 5.94 5.74
31.6 6.05 5.72
100 6.06 5.80
316 6.07 5.74
1000 6.08 5.74
Moisture:
Test item concentration Moisture [%] of DW
[mg/kg SDW] [g /100 g SDW]
day 0 day 14
Control 24.6 23.1
3.16 24.9 23.9
10 24.5 23.0
31.6 24.6 23.1
100 24.6 22.9
316 24.8 24.2
1000 25.1 23.7
Details on test conditions:
TEST SYSTEM
- Test containers Glass vessels (volume 1.5 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 per replicate
- No. of organisms per container (treatment): 10 per replicate
- No. of replicates per treatment group: 4
- No. of replicates per control: 4

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)
- 0.40 % 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 (17 kg) was pre-moistened with 1826 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 ± 83.5

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: factor √10
- Justification for using less concentrations than requested by guideline:
- Range finding study
A preliminary range finding test was performed with 3 test item concentrations.
2 replicates without the addition of food with 10 earthworms each.

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 -11
10 0 -13
100 0 -21
1000 80 -42
Nominal and measured concentrations:
Nominal: 3.16 – 10 – 31.6 – 100 – 316 – 1000 mg/kg soil dry weight
Reference substance (positive control):
yes
Remarks:
2-Chloroacetamide
Key result
Duration:
14 d
Dose descriptor:
EC50
Effect conc.:
754 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: body weight
Remarks on result:
other: CI (95%) = 394 - 1401 mg/kg soil DW
Key result
Duration:
14 d
Dose descriptor:
LC50
Effect conc.:
1 018 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: CI (95%) = 756 - 1703 mg/kg soil DW
Key result
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
Key result
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
316 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Mortality, behaviour, pathological symptoms
Key result
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: Change of body weight
Key result
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
316 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Change of body weight
Key result
Duration:
14 d
Dose descriptor:
LC10
Effect conc.:
309 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: CI (95%) = 171 - 424 mg/kg soil DW
Key result
Duration:
14 d
Dose descriptor:
other: LC20
Effect conc.:
466 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: CI (95%) = 315 - 618 mg/kg soil DW
Key result
Duration:
14 d
Dose descriptor:
EC10
Effect conc.:
171 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: body weight
Remarks on result:
other: CI (95%) = 104 - 281 mg/kg soil DW
Key result
Duration:
14 d
Dose descriptor:
other:
Remarks:
EC20
Effect conc.:
284 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: body weight
Remarks on result:
other: CI (95%) = 174 - 466 mg/kg soil DW
Details on results:
- Mortality at end of exposure period:
A statistically significant mortality was observed in the test item concentration 1000 mg/kg soil dry weight compared to the control. Whereas no statistically significant mortalities were observed in the test item concentrations 3.16 to 31.6 mg/kg soil dry weight. In the test item concentrations 100 mg/kg soil dry weight and 316 mg/kg soil dry weight low mortalities were observed after 14 days of exposure.
- Total mass of adults at beginning of test:
Control: 18.50 g
3.16 mg/kg soil DW: 18.60 g
10 mg/kg soil DW: 18.60 g
31.6 mg/kg soil DW: 18.60 g
100 mg/kg soil DW: 18.60 g
316 mg/kg soil DW: 18.60 g
1000 mg/kg soil DW: 18.60 g
- Changes in body weight of live adults (% of initial weight) at end of exposure period:
In terms of body weight loss, the NOEC was determined to be 316 mg/kg soil dry weight and LOEC was determined to be 1000 mg/kg soil dry weight.
- Morphological abnormalities:
No pathological symptoms or changes in the earthworm behaviour were observed in the control as well as in the test item concentrations 3.16 to 100 mg/kg soil dry weight after 7 and 14 days of exposure. In the test item concentration 316 mg/kg soil dry weight and 1000 mg/kg soil dry weight, significant changes in earthworm behaviour were observed after 7 and 14 days.
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:
LC/EC values
The LCx and ECx values were determined by Probit analysis using linear max. likelihood regression and by Analysis of Variance and Test for Lack of Fit for the 3-param. Normal CDF, respectively.

NOEC, LOEC and statistical analyses
The NOEC/LOEC for mortality was determined using a Multiple Sequentially-rejective Fisher Test After Bonferroni-Holm for determination of statistically significant differences in mortality compared to the control. When running the test, a Qualitative Trend Analysis by Contrasts and a Tarone’s Test Procedure were done first.
Statistically significant differences in biomass compared to the control were determined using a Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm. When running the test, a Shapiro-Wilk’s Test on Normal Distribution and a Levene’s Test on Variance Homogeneity (with Residuals) 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

MV

1

2

3

4

MV

Control

0

0

0

0

0

0

0

0

0

0

3.16

0

0

0

0

0

0

0

0

0

0

10

0

0

0

0

0

0

0

0

0

0

31.6

0

0

0

0

0

0

0

0

0

0

100

0

0

0

0

0

10

0

0

0

2.5

316

0

0

0

10

2.5

10

0

0

10

5

1000

10

10

20

10

12.5

60

40

30

80

52.5

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

Day 7

Control

A)

10/10

10/10

10/10

10/10

3.16

A)

10/10

10/10

10/10

10/10

10

A)

10/10

10/10

10/10

10/10

31.6

A)

10/10

10/10

10/10

10/10

100

A)

10/10

10/10

10/10

10/10

316

A)

D)

H)

9/10

1/10

-

10/10

-

-

10/10

-

-

9/10

-

1/10

1000

A)

8/10

9/10

8/10

9/10

D)

H)

1/10

1/10

-

1/10

-

2/10

-

1/10

 

Day 14

Control

A)

10/10

10/10

10/10

10/10

3.16

A)

10/10

10/10

10/10

10/10

10

A)

10/10

10/10

10/10

10/10

31.6

A)

10/10

10/10

10/10

10/10

100

A)

H)

9/10

1/10

10/10

-

10/10

-

10/10

-

316

A)

C)

D)

H)

6/10

1/10

3/10

1/10

3/10

-

7/10

-

10/10

-

-

-

6/10

-

3/10

1/10

1000

A)

3/10

4/10

4/10

2/10

D)

1/10

2/10

3/10

-

H)

6/10

4/10

3/10

8/10

A) no obvious pathological symptoms                      C) no negative phototactical reaction       

D) spontaneous segmentation and separation                              H) dead earthworm

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

0.46 ± 0.04

0.41 ± 0.04

10.9

3.16

0.46 ± 0.04

0.40 ± 0.04

13.0

No

10

0.47 ± 0.04

0.41 ± 0.05

12.8

No

31.6

0.46 ± 0.04

0.40 ± 0.04

13.0

No

100

0.46 ± 0.04

0.38 ± 0.05

17.4

No

316

0.46 ± 0.04

0.34 ± 0.11

26.1

No

1000

0.47 ± 0.04

0.33 ± 0.11

29.8

Yes

MV = Mean value           SD = Standard deviation

Validity criteria fulfilled:
yes
Conclusions:
No statistically significant mortalities were observed in the test item concentrations 3.16 to 316 mg/kg soil dry weight compared to the control, whereas a statistically significant mortality was observed in the test item concentration of 1000 mg/kg soil dry weight after 14 days of exposure. Consequently, the NOEC was determined to be 316 mg/kg soil dry weight and LOEC was determined to be 1000 mg/kg soil dry weight.
In terms of body weight loss, the NOEC was determined to be 316 mg/kg soil dry weight and LOEC was determined to be 1000 mg/kg soil dry weight.
Executive summary:

The acute effects of EDTA-CuK2 on the earthworm Eisenia fetida were determined according to the OECD Guideline 207. The study was conducted under static conditions for 14 days with the test item concentrations 3.16 – 10 – 31.6 – 100 – 316 and 1000 mg/kg soil dry weight mixed into artificial soil containing 10 % peat. Artificial soil without test item was used as control. 40 test organisms with an individual weight between 0.40 - 0.54 g, divided into four replicates, were tested per test item concentration and control.

No statistically significant mortalities were observed in the test item concentrations 3.16 to 316 mg/kg soil dry weight compared to the control, whereas a statistically significant mortality was observed in the test item concentration of 1000 mg/kg soil dry weight after 14 days of exposure. Consequently, the NOEC was determined to be 316 mg/kg soil dry weight and LOEC was determined to be 1000 mg/kg soil dry weight.

In terms of body weight loss, the NOEC was determined to be 316 mg/kg soil dry weight and LOEC was determined to be 1000 mg/kg soil dry weight.

All validity criteria of the test guideline were fulfilled.

 

Summary of all Effects

Effects

EDTA-CuK2

[mg/kg soil dry weight]

LOEC

Mortality, behaviour, pathological symptoms

1000

NOEC

Mortality, behaviour, pathological symptoms

316

LOEC

Change of body weight

1000

NOEC

Change of body weight

316

LC10mortality(confidence interval)

LC20mortality(confidence interval)

LC50mortality(confidence interval)

309 (171 - 424)                                 

466 (315 - 618)

 1018* (756 – 1703*)

EC10body weight(confidence interval)

EC20body weight(confidence interval)

EC50body weight(confidence interval) 

171 (104 - 281)

284 (174 - 466)

 754 (394 – 1401*) 

*: values were extrapolated

Description of key information

One acute toxicity study with earthworms is available for EDTA-CuK2 (Winkelmann, 2019). The EC50 for weight was determined to be 754 (394 - 1401) mg/kg dw. The LC50 for mortality is 1018 (756 - 1703) mg/kg dw.       

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:
754 mg/kg soil dw

Additional information

The acute effects of EDTA-CuK2 on the earthworm Eisenia fetida were determined according to the OECD Guideline 207. The study was conducted under static conditions for 14 days with the test item concentrations 3.16 – 10 – 31.6 – 100 – 316 and 1000 mg/kg soil dry weight mixed into artificial soil containing 10 % peat. Artificial soil without test item was used as control. 40 test organisms with an individual weight between 0.40 - 0.54 g, divided into four replicates, were tested per test item concentration and control.

Nostatistically significant mortalities were observed in the test item concentrations 3.16 to 316 mg/kg soil dry weightcompared to the control, whereasa statistically significant mortality was observed in the test item concentration of 1000 mg/kg soil dry weight after 14 days of exposure.Consequently, the NOEC was determined to be 316 mg/kg soil dry weight and LOEC was determined to be 1000 mg/kg soil dry weight.

In terms of body weight loss, the NOEC was determined to be 316 mg/kg soil dry weight and LOEC was determined to be 1000 mg/kg soil dry weight.

All validity criteria of the test guideline were fulfilled.