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Ecotoxicological information

Toxicity to soil macroorganisms except arthropods

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Reference
Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 Jan - 07 Jun 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Analytical monitoring:
no
Vehicle:
no
Details on preparation and application of test substrate:
- Method of mixing into soil: test item was mixed in quartz sand (10% of the quartz sand from the total amount of the artificial soil); these mixtures were used to achieve the required test item concentrations; test item was incorporated into the remainder of the artificial soil by thoroughly mixing in the respective test item/quartz sand mixtures for 5 minutes and was placed into the test vessels; 500 g dw (dry weight) was used per test vessel.
- Controls: yes (untreated quarz sand added to artificial soil)
Test organisms (species):
Eisenia andrei
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: Earthworm
- Source: Ibacon GmbH, Arheilger Weg 17, D-64380 Rossdorf, Germany
- Age at test initiation (mean and range, SD): at least two months, but not more than one year old, however, the age of individuals did not differ by more than four weeks
- Weight at test initiation (mean and range, SD): fresh weight (FW) between 250 and 600 mg (adult worms)

ACCLIMATION
- Acclimation period: selected worms were acclimatised at least 24 hours before starting the test.
- Acclimation conditions: in artificial soil under test conditions
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
56 d
Test temperature:
18.8–20.8 °C
pH:
5.7–5.8 (soil pH day 0)
6.3–6.5 (soil pH day 56)
Moisture:
49.7–51.4% of the WHCmax (Day 0)
59.4–68.7% of the WHCmax (Day 56)
Details on test conditions:
TEST SYSTEM
- Test container (material, size): Bellaplast containers (Company Kastelplast, 55120 Mainz, Germany) inert (non-toxic) plastic (Polystyrol), 11 x 15.5 cm (= 170.5 cm²) and a height of 6 cm; covered with a transparent and perforated lid.
- Amount of soil or substrate: 500 g dw per test vessel
- No. of organisms per container (treatment): 10 per test vessel
- No. of replicates per treatment group: 4
- No. of replicates per control: 8

SOURCE AND PROPERTIES OF SUBSTRATE
- Collection procedures: artificial soil
- Composition: According to the guideline OECD 222: 5% Sphagnum peat, 20% Kaolin clay, 74–75% Quartz sand and 0.3–1% Calcium carbonate (CaCO3).
- Other information on artificial soil: due to the test item (log Pow≥2), considering the influence on bioavailability, 5% instead of 10% peat were used (EPPO 2003); The air-dried peat was shredded in a chaff-cutter. Afterwards, all parts of the artificial soil were mixed homogeneously. The pH value was adjusted to 6±0.5 using calcium carbonate. The moisture content was adjusted to 40–60% of WHCmax using deionised water. The water content of the test substrate was checked weekly by reweighing the test vessels and losses of water were compensated.

OTHER TEST CONDITIONS
- Photoperiod: 16:8 h light:dark
- Light intensity: 437–642 lux

EFFECT PARAMETERS MEASURED: Mortality and biomass after 28 days; reproduction after 56 days

VEHICLE CONTROL PERFORMED: no

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
Range finding study (non-GLP)
- Test concentrations: 0.1, 1.0, 10.0, 100 and 1000 mg test item/kg artificial soil dry weight
- Results used to determine the conditions for the definitive study: No mortality was observed in the control and at the concentrations of 1.0, 10, 100 and 1000 mg test item/kg soil (dw). At the concentration of 0.1 mg test item/kg soil (dw) 10% mortality was observed. No clear dose response and inhibition in number of cocoons was observed.
Nominal and measured concentrations:
Control, 62.5, 125, 250, 500 and 1000 mg test item/kg artificial soil dry weight (nominal)
Reference substance (positive control):
yes
Remarks:
boric acid
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
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:
other: Biomass
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
56 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
- Mortality at end of exposure period: No mortality was observed throughout all test vessels after 28 days.
- Other biological observations: See biological results summarized within the Tables 1-3 in section any other information on results incl. tables
Results with reference substance (positive control):
Boric acid was tested in parallel as the toxic reference item as recommended by the ISO 11268-2 Part 2 (2011) guideline. It was used at a concentration of 500 mg boric acid/kg soil (dw). After 28 days no mortality was observed whereat a clear effect on reproduction (no offspring) was observed after 56 days. This observed effect is according to the ISO guideline, i.e. observed effects on reproduction should be between 400 and 600 mg boric acid/kg soil (dw). Hence, acceptable sensitivity of the test system is assured.
Reported statistics and error estimates:
Biomass: Data were checked for normality by R/s test procedure and for homogeneity by Cochran’s test. Treatment means were compared by ANOVA followed by Dunnett’s t-test (p ≤0.05, two-sided) and tested for statistically significant differences compared to the control (Sachs 1982).
Reproduction: Data were checked for normality by R/s test procedure and for homogeneity by Cochran’s test. Treatment means were compared by ANOVA followed by Dunnett’s t-test (p ≤0.05, one-sided) and tested for statistically significant differences compared to the control (Sachs 1982).
The statistical software package ToxRat Professional 3.2.1 was used for these calculations.

Validity criteria:

Mortality of the adults in the control = 0% (required: ≤10%);

Mean number of juveniles in the control = 297.3 (required: ≥30)

CV for the number of juveniles in the control = 9.6 (required: ≤30%)

Conclusion:

The effects of the test item on mortality, biomass development, morphological changes and reproduction to the earthworm Eisenia andrei in artificial soil were tested in the laboratory. At concentrations up to and including 1000 mg test item/kg soil dry weight the test item had no adverse effects on either mortality, biomass development, morphological changes and reproduction to the test species.

Table 1: Results of the main test-effects on mortality, biomass and reproduction of Eisenia andrei.

Test item

Test Item name

Test system

Eisenia andrei

Exposure

OECD artificial soil

Concentration [mg test item/kg soil (dw)]

Adult mortality [%]

Biomass [Mean change in % of the initial weight per replicate]

Number of Juveniles [% of Control]

Control

0.0

90.0

-

62.5

0.0

93.4

94.5

125

0.0

99.4

98.2

250

0.0

99.1

98.7

500

0.0

95.8

90.1

1000.0

0.0

97.6

94.7

Reference

0.0

72.3

0.0

NOEC [mg test item/kg soil (dw)]

≥1000

≥1000

≥1000

LOEC [mg test item/kg soil (dw)]

>1000

>1000

>1000

EC50 [mg test item/kg soil (dw)]

-

-

>1000

- not applicable

Table 2: Main test: Adult biomass

Concentration [mg test item/kg soil (dw)]

Biomass (mg fresh weight)

Mean change in % of the initial weight per replicate

 

Day 0

Day 28

Day 28

Control

370.5 ± 21.4

702.3 ± 23.0

90.0

62.5

370.5 ± 19.6

714.9 ± 14.7

93.4

125

366.6 ± 23.5

729.7 ± 22.4

99.4

250

371.2 ± 20.2

738.2 ± 17.3

99.1

500

369.4 ± 19.8

722.7 ± 26.8

95.8

1000

373.6 ± 17.5

737.9 ± 37.9

97.6

Average live weight (mg) per surviving adult worm at the beginning of the test and after 28 days (n = 80 for the controls; n = 40 for test concentrations; n = 80 for the reference item). Data are given as absolute value and in mean percent of the initial weight change per replicate.

Table 3: Main test: Reproduction

Concentration [mg test item/kg soil (dw)]

Number of Juveniles per Treatment (mean ± sd)

[% of Control]

Control

297.3 ± 28.5

100.0

62.5

280.8 ± 41.5

94.5

125

291.8 ± 29.3

98.2

250

293.3 ± 44.9

98.7

500

268.0 ± 11.4

90.1

1000

281.5 ± 60.1

94.7

Reproduction (given as mean number of juveniles ± standard deviation and as % of the controls) in the treated test vessels.

Validity criteria fulfilled:
yes

Description of key information

NOEC (28 d) ≥ 1000 mg test item/kg dry weight soil (mortality and biomass, Eisenia andrei, OECD 222)

NOEC (56 d) ≥ 1000 mg test item/kg dry weight soil (reproduction, Eisenia andrei, OECD 222)

EC50 (56 d) > 1000 mg test item/kg dry weight soil (reproduction, Eisenia andrei, OECD 222)

Key value for chemical safety assessment

Additional information

One experimental study is available investigating the toxicological effects of the test item (CAS 16260-09-6) to earthworms (ECT, 2017). The study was performed according to OECD guideline 222 under GLP conditions. Adult Eisenia andrei (4 x 10 animals per concentration, the fresh weight was between 250 and 600 mg) were exposed for 28 days in an artificial soil to the nominal concentrations of 62.5, 125, 250, 500 and 1000 mg test item/kg artificial soil dry weight. The artificial soil comprised 5% sphagnum peat, 20% kaolin clay, 74–75% Quartz sand and 0.3 - 1% calcium carbonate. No morphological and behavioral effects were observed. Related to weight alterations and symptoms, the no-observed-effect-concentration (NOEC) was ≥ 1000 mg test item/kg dry weight soil, the lowest-observed-effect-concentration (LOEC) > 1000 mg test item/kg dry weight soil for all parameters after 28 days for mortality and biomass. No mortality was recorded resulting in a EC50 (28 d) of > 1000 mg test item/kg dry weight soil. For reproduction the no observed effect concentration (NOEC) was observed at ≥ 1000 mg test item/kg dry weight soil and the EC50 at > 1000 mg test item/kg dry weight soil after 56 days.