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Toxicity to soil macroorganisms except arthropods

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Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
ISO 11268-2 (Effects of Pollutants on Earthworms. 2. Determination of Effects on Reproduction)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: BBA Guideline Teil VI, 2-2
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Details on preparation and application of test substrate:
-Preparation: On the day of application, the spray solutions for the toxic standard and the Karmex 80 WG treatments were prepared in the concentrations of 5.3, 10.7, 26.7, 133.3 and 266.7 mg/kg artificial soil. The spray solutions were prepared immediately prior to application
- Application: The deionized water (for the control), the test substance solution and the toxic standard solution were applied, using an automatic spraying-chamber simulating a field application. The spraying-chamber was calibrated before application by adjusting the spraying pressure, application speed and type of nozzle to provide an output of 400 L/ha. The permitted range of quantity applied was 4 mg/cm2 f 10 % which is equal to a dosage of 400 L/ha ± 10 %

Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: Earthworm
- Source: Healthy laboratory rearing stock of the testing facility
- Age at test initiation: About two months old with clitellum
- Weight at test initiation: about 400 mg

CULTURING CONDITIONS
The earthworms were cultured at 20°C ± 2°C, under continuous light (approx. 500 Lux) in a climate-controlled chamber. They were kept in plastic containers,
27 cm by 35 cm by 23 cm deep, in a horse dung substrate. Each container holds approximately 200 earthworms in different developmental stages

ACCLIMATION
The worms were acclimatized by placing them in untreated test medium under environmental test conditions for 1 day prior to the start of this study
Study type:
laboratory study
Substrate type:
artificial soil
Total exposure duration:
56 d
Remarks:
28 days (mortality and effect on biomass) + 28 days (fertility)
Test temperature:
20°C ±2°C
pH:
6.5
Moisture:
30 g/100g oven-dry soil
Details on test conditions:
TEST SYSTEM
- Test container (material, size): White plastic vessels (BELLAPLAST, 17 cm x 12.5 cm x 6 cm)
- Amount of soil or substrate: Vessels were filled with 767.7 g moist artificial soil (corresponding to 600 g dry artificial soil) to a height of 4 - 5 cm. The surface covered about 200 cm
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 4

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
-Artificial soil in accordance with OECD guideline 207: 10% sphagnum peat, 20% kaolinite clay, 69% industrial quartz sand and 1% calcium carbonate (% in terms of dry weight). pH was 6.5 and moisture content 30 g/100 g

OTHER TEST CONDITIONS
- TOC content: 36.5 g C/kg dry artificial soil
- Water holding capacity: 55.9 mL/100g
- Photoperiod: 16:8 h photoperiod (long day conditions).

EFFECT PARAMETERS MEASURED: Mortality, growth and reproduction
After 28 days the number of surviving earthworms, the body weight, the water content and the pH of the artificial soil were recorded.
After the 4 week assessment, the artificial soil substrate - cocoons included -was refilled into the test containers, to allow hatching and developing of juveniles. About 5 g of food was mixed into the artificial soil and the test containers were incubated for an additional period of 28 days under test conditions. After 56 days the test was terminated and the number of offspring was recorded

Nominal and measured concentrations:
Nominal
5.3, 10.7, 26.7, 133.3 and 266.7 mg a.i./kg artificial soil
Reference substance (positive control):
yes
Remarks:
Carbendazim (360 g/L)
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
21.3 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: corresponding to 10 kg Karmex 80 WG/ha
Duration:
56 d
Dose descriptor:
LOEC
Effect conc.:
53.3
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 25 kg Karmex 80 WG/ha
Results with reference substance (positive control):
In the toxic standard group a significant decrease of the body weight and of juveniles hatched after 56 days compared with the control was observed (multiple test procedure according to Dunnett, a = 0.05)
Reported statistics and error estimates:
Statistical analysis was calculated with the EASY ASSAY Multiple Testing program at the computer system of the testing facility. After proving the homogeneity of variance, a multiple t-test (Dunnett-test) was conducted to compare the control with the independent test substance groups for significant or non-significant differences

- No adult earthworm mortality was observed at any treatment including the controls, the Karmex 80 WG treatments and the toxic standard

- No statistically significant reduction of body weight could be observed in the treatment groups compared to the control

- Concerning the number of juveniles no significant difference was calculated at the treatment rates of 5.3 mg a.i./kg and 10.7 mg a.i./kg of Karmex 80 WG compared with the control (multiple test procedure according to Dunnett, a 0.05, one sided smaller). Reduction of Earthworm reproduction was calculated to be statistically significant at the treatment groups of 26.7 mg a.i./kg, 133.3 mg a.i./kg, 266.7 mg a.i./kg and the toxic standard group, compared to the control

Table 1 Effect data

 

 

Test Substance Concentration

Karmex 80 WG

[mg a.i./kg artificial soil]

Mortality after 28 d

Earthworm body weight
(mg)

Reproduction capacity after 56 days

 

 

No. of dead earthworms

Percentage (n=40)

Mean after 28 days

 

Mean number of juvenile worms of exposure

Control

0

0

363

151

5.3

0

0

361

136

10.7

0

0

393

142

26.7

0

0

401

126*

133.33

0

0

352

100*

266.7

0

0

353

98*

Toxic standard

0

0

319*

77*

* Statistically significantly different from the mean control value (multiple test procedure according to Dunnett, a 0.05, one sided smaller)

Table 2 Validity criteria

 

Fulfilled

Not fulfilled

Mortality of control animals < 10%

x

 

The loss in the biomass of the worms in the control did not exceed 20 %

x

 

The average number of juvenile earthworms in the control group per vessel was > 30

x

 

The coefficient of variance of reproduction in the control was <50 %,

x

 

The reproduction rate by the toxic standard should be lower than the reproduction rate by the control

x

 

Executive summary:

A chronic toxicity test was conducted in accordance with prevailing guidelines ( BBA Guideline Teil VI, 2-2 and ISO Guideline 11268-2) with GLP, however, the test concentrations were not confirmed by chemical analysis.

The NOEC for effects on biomass and reproduction was determined to be 21.3 mg a.i./kg dry artificial soil corresponding to 10 kg Karmex 80 WG/ha. The LOEC for effects on biomass was determined to be 53.3 mg a.i./kg dry artificial soil corresponding to 25 kg Karmex 80 WG/ha. These results indicate that the test item is in all probability not harmful to soild macroorganisms following chronic exposure.

Endpoint:
toxicity to soil macroorganisms except arthropods: short-term
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 207 (Earthworm, Acute Toxicity Tests)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on preparation and application of test substrate:
5 g of the test substance was weighed, added to 500 ml of deionised water and stirred on a magnetic stirrer for 1/2 hour. The concentrations for this study were prepared by mixing equivalent parts of this stock solution into the test substrate thoroughly with a domestic mixer. At the same time, the rest of the deionised water required was mixed into the test substrate in each test container.
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: earthworm
- Source: Prof Graff, Forschungsanstalt für Landwirtschaft, D-3300-Braunschweig
- Age at test initiation: More than two months old worms
- Weight at test initiation: exceeding 400 mg (mean weight of 450 mg)

ACCLIMATION
- Acclimation period: 14 days
- Acclimation conditions (same as test or not): Shallow glass trays, breeding medium (70% by weight of natural soil, 25% peat and 5% straw) at 22 ± 2°C with 12/12h light/dark cycle.
Study type:
laboratory study
Substrate type:
artificial soil
Total exposure duration:
14 d
Test temperature:
20°C ±1°C
pH:
pH: 6.0 ± 0.5
Moisture:
Moisture content: 26.3 – 30.7 g/100g oven-dry soil
Details on test conditions:
TEST SYSTEM
- Test container (material, size): 1.5 L preserving jars, covered with glass lids
- Amount of soil or substrate: 0.725 kg wet weight
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 4

PROPERTIES OF SUBSTRATE:
Artificial soil in accordance with OECD guideline 207: 10% sphagnum peat, 20% kaolinite clay, 69% industrial quartz sand and 1% calcium carbonate (% in terms of dry weight)

OTHER TEST CONDITIONS
- Photoperiod: 12/12 h light/dark cycle
- Light intensity: 400-800 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality (examined at 7 and 14 days)
Nominal and measured concentrations:
Nominal: 56, 100, 178, 316, 562 and 1000 mg/kg dry weight artificial soil
Reference substance (positive control):
yes
Remarks:
Chloroacetamide
Duration:
14 d
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
178 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: weight alteration and symptoms
Duration:
14 d
Dose descriptor:
LOEC
Effect conc.:
316 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: weight alteration and symptoms
Details on results:
- Mortality at end of exposure period: All animals in the control vessels survived. One earthworm was found to be dead at a concentration rate of 100 mg/kg soil dry weight after seven days. Mortality was not regarded test substance related, but as a limit for natural mortality
- Other biological observations: No other observations differentiating organisms in tests and controls were realized
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: LC50 (14 days) of 13.3 mg/kg dry weight substrate (95% confidence limits 12.4 - 14.3 mg / kg)
- Other: within the concentration range normally determined in international ring studies
Reported statistics and error estimates:
Weight alterations of the test organisms were statistically evaluated by the U-Test of Wilcoxon, Mann & Whitney (L. SACHS: Angewandte Statistik, Springer Verlag 1978; Probability level P = 0.05, two sided)

Table 1:              Mortality data of the test substance

Test Substance

Concentration
(nominal)1

[mg/kg artificial soil]

Mortality

Number

             7 d                               14 d

Percentage

             7 d                               14 d

control

0

0

0

0

56

0

0

0

0

100

1

1

2.5

2.5

178

0

0

0

0

316

0

0

0

0

562

0

0

0

0

1000

0

0

0

0

Temperature [°C]

20

 

pH

6.0

 

Moisture content

30.7g/100g

 

 

 Table 2:  Validity criteria for acute earthworm test according to OECD 207

 

Fulfilled

Not fulfilled

Mortality of control animals < 10%

x

 

Executive summary:

An acute toxicity test was conducted in accordance with the prevailing OECD testing guidelines (OECD 207), with GLP. However, the test concentrations were not confirmed by chemical analysis.

The LC50(14 days) determined for Diuron WP 80 was > 1000 mg/kg dry weight substrate. Related to weight alteration and symptoms the no-observed-effect concentration (NOEC) was 178 mg/kg dry weight substrate. These values correspond to an LC50(14d) of > 800 mg Diuron/kg and a NOEC of 142.4 mg Diuron/kg.

The calculated LC50(14d) of > 800 mg/kg indicates that Diuron is not considered to be toxic to earthworms under test conditions

Description of key information

Diuron is not considered to be toxic to eathworms under test conditions

Key value for chemical safety assessment

Additional information

One chronic toxicity test was conducted in accordance with prevailing guidelines ( BBA Guideline Teil VI, 2-2 and ISO Guideline 11268-2) with GLP, however, the test concentrations were not confirmed by chemical analysis (Stäbler, 2001).The NOEC for effects on biomass and reproduction was determined to be 21.3 mg a.i./kg dry artificial soil corresponding to 10 kg Karmex 80 WG/ha. The LOEC for effects on biomass was determined to be 53.3 mg a.i./kg dry artificial soil corresponding to 25 kg Karmex 80 WG/ha. These results indicate that the test item is in all probability not harmful to earthworms following chronic exposure.Furthermore, an acute toxicity test was conducted in accordance with the prevailing OECD testing guidelines (OECD 207), with GLP. This derived a LC50(14 days) for Diuron WP 80 of > 800 mg Diuron/kg and a NOEC of 142.4 mg Diuron/kg (Heimbach, 1993).

Discussion on endocrine disruption:

As at the current stage there is still no agreed approach and guidance on how to evaluate endocrine disruption in various EU legislations the OECD Conceptual framework for Testing and Assessment of Endocrine Disruptors might be used. Comparing available data with the Framework`s level concept allowed to rank Stäbler (2001) in OECD level 1, which is the lowest level and such studies do not allow to conclude on endocrine properties of diuron. This study assessed effects of Diuron exposure on reproduction and growth of Eisenia foetida according to GLP standard and a BBA guideline part VI, 2-2 / ISO guideline 11268-2, which may be understood as a precursor to OECD test guideline 222. As there is no such study among those listed in level 2 to 5 this study may only be ranked as level 1. This study reports of a reduced number of juveniles when exposed to a test item concentration which is at least 5-fold higher than the field application rate and, thus, was performed under unrealistic test conditions. In conclusion the study result are not adequate for decision on endocrine properties of diuron. See also the document on the ad hoc assessment on endicrine disruption properties of diuron attached in chapter 13 of IUCLID.