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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:
07 Feb 2017 to 05 Apr 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:
July, 2016
Qualifier:
according to
Guideline:
ISO 11268-2 (Effects of Pollutants on Earthworms. 2. Determination of Effects on Reproduction)
Version / remarks:
2012
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Name of test material (as cited in study report): ZDEC
- Batch No.: 70100804
- Purity: 99.2%
- Zn. content: 18.1%
- Date of analysis: January 20, 2017
- Appearance: Solid white to off white powder or Granules
- Solubility (water): 1.06 mg/L
- Storage conditions: at 20 ± 5 °C, in the dark
Analytical monitoring:
no
Vehicle:
yes
Remarks:
quartz sand
Details on preparation and application of test substrate:
The test substance was weighed separately for each concentration using an analytical balance and 20 g of fine quartz sand was added to each weighing. After mixing with a spoon to reach a homogeneous distribution of the test item within the sand the mixture was added to artificial soil equivalent to 2080 g dry weight. There were no significant deviations to the nominal target concentration (< 5%) after substance preparation. While mixing the artificial soil in a laboratory mixer for approximately 5 min the soil of each treatment group was moistened with deionised water. Each group was treated in one batch (two in the control) which was then split into the replicates. The application order was as follows: 1. control, 2. test item (from low to high concentrations).
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: Earthworm
- Source: In-house culture
- Age at test initiation: Adult worms, approximately 9 to 10 months old, with well-developed clitellum, age range between test individuals not differing by more than 4 weeks
- Weight: 311 to 598 mg
- Origin: Bred under standardised conditions at the test laboratories in a breeding medium of cattle manure, peat, sand, calcium carbonate and straw, fed with cattle manure, stored at room temperature.

ACCLIMATION
- Acclimation period: 1 day, in artificial soil, under test conditions (see 'Details on test conditions').
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
8 wk
Post exposure observation period:
- Exposure time adult worms: 4 weeks
- Exposure time of offspring: 4 weeks (additional 28 days)
Test temperature:
18 - 22 °C
pH:
- At experimental start: 5.7
- At experimental end: 6.3 - 6.4
Moisture:
- At experimental start: 28.6% to 30.5% (52.0% to 55.5% of the maximum water holding capacity, i.e. within the recommended range 40-60 % of the total water holding capacity).
- At experimental end: 30.6% to 32.4% (55.6% to 58.9% of the maximum water holding capacity).
Details on test conditions:
TEST SYSTEM
- Test container: Plastic boxes (18.3 cm x 13.6 cm x 6 cm, tapered towards the bottom, with a soil surface of approximately 16.5 cm x 11.5 cm = 189.75 cm²) with perforated transparent lids to enable exchange of air, to minimise evaporation from the artificial soil, and to prevent the worms from escaping.
- Amount of soil or substrate: Each container was filled with 643.6 g of the prepared soil (500 g dry weight plus deionised water). The height of the soil layer in the containers was approximately 5 cm.
- No. of individuals per container: 10
- No. of replicates per treatment group: 4
- No. of replicates per vehicle control: 8
- Food: Finely ground cattle manure was used as food. 5 g/container was scattered on the soil surface at day 1 after application and was moistened with 5 g deionised water; 5 g/container (moistened with 2 - 3 g deionised water) was added each week for the first 4 weeks of the experiment, when the food of the previous week had almost been consumed. If the food was not quite fully consumed, the added amount of food was adjusted to replace the visually estimated consumption. Four weeks after application, the food was mixed into the substrate following removal of the adult worms.
- Introduction of individuals: All worms were rinsed with tap water, dried with dry paper towels, weighed individually and randomly assigned to batches of 10 worms. The different batches were sorted into four classes on the basis of the total weight and one batch of each weight class was assigned to each treatment group (two batches for the control) to ensure weights were homogeneous. The earthworms were placed on the surface of the artificial soil after application.

SOURCE AND PROPERTIES OF SUBSTRATE
- % peat: 10% Sphagnum-peat, air-dried and finely ground (<2 mm, with no visible plant remains); (Floragard, Vertriebs GmbH für Gartenbau, 26138 Oldenburg, Germany)
- % sand: 69.65% fine quartz-sand (F34) containing more than 50% by mass of particle size 0.05 mm to 0.2 mm; (Quarzwerke Frechen, Postfach 1780, 50207 Frechen, Germany)
- % clay: 20% Kaolin clay (Kaolinite content >30%) (Erbslöh, 65558 Lohrheim, Germany)
- Adjusted pH: yes, 0.35% Calcium carbonate (CaCO3) was added to adjust pH to 6.0 ± 0.5 (Merck, 64293 Darmstadt, Germany).
- Maximum water holding capacity: 55% of the dry weight of artificial soil

OTHER TEST CONDITIONS
- Photoperiod: 16 h light : 8 h dark
- Light intensity: 400 - 800 lux
- Water content maintenance: Once a week the water content of the soil was checked by weighing each container and evaporated water was replenished. In this way, it was ensured that the difference in water content between experimental start and end was less than 10%.
- pH: pH was determined at the start and end of the test for each treatment group. The pH range at experimental start was 5.7 and thus did not deviate from the values given by the guidelines (6.0 ± 0.5). The pH range at experimental end was 6.3 to 6.4.
- Test environment: Controlled environment room, in a ventilated area.

TEST COURSE
- Exposure Time of Adult Worms: After 4 weeks, the artificial soil was transferred to a tray and adult worms were counted, removed and weighed per replicate after being rinsed under tap water and dried on paper towels. Missing earthworms and earthworms that failed to respond to gentle stimulation were considered to be dead. The remaining soil (without the adult worms) was then returned to the respective test containers.
- Removal of Juveniles: Juveniles were removed by placing the test units in a water bath at 50 - 60 °C and counting all emerging worms. In addition the soil of each container was emptied out onto a tray and checked visually for any remaining juvenile worms.

EFFECT PARAMETERS MEASURED
- Mortality: Number of dead adult earthworms at day 28 after application (including any missing ones)
- Behavioural and Morphological Abnormalities: Number of affected adult earthworms (e.g. lack of movement, rigidity) at day 28 after application
- Food Given: Cumulative amount of food added to each test container during the test period
- Mean Body Weight: Body weights were determined at start (day 0) and 28 days after application
- Reproduction: Number of juveniles 56 days after application

VEHICLE CONTROL PERFORMED: yes (the same amount of untreated quartz sand as in the test item treated groups was added and moistened with deionised water)
Nominal and measured concentrations:
- Nominal concentrations: 0 (control), 1, 2, 4, 8 and 16 mg test item/kg soil dw
Reference substance (positive control):
yes
Remarks:
Carbendazim (600 g/L nominal)
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
16 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
16 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
16 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
based on body weight
Details on results:
- Mortality: No mortality was observed in any treatment group.
- Body weights of adult worms: The body weight changes in the test item treated groups were not statistically significantly different compared to the control up to and including the highest test concentration of 16 mg test item/kg soil. See 'Any other information on materials and methods incl. tables'.
- Reproduction: The reproduction rates were not statistically significantly different compared to the control up to and including the highest test concentration. See 'Any other information on materials and methods incl. tables'.
- Feeding activity: In all treatment groups, food was consumed. The results show that the turnover of biomass of those earthworms exposed to the different rates of the test item was comparable to the control.
- Behavioural Abnormalities: No behavioural abnormalities were observed and all worms burrowed into the soil within 15 minutes after introduction.
Results with reference substance (positive control):
The reference item test (dose response) is performed at least once a year at the test facility as a means of ensuring that the laboratory test conditions are adequate and to verify that the response of the test organisms does not change significantly over time. The GLP conducted experiment was performed from June to August 2016. The 28-d NOEC based on mortality and weight was determined to be ≥3.00 mg a.i./kg soil dw. The 56-d NOEC and 56-d LOEC based on reproduction was determined to be 1.44 and 2.08 mg a.i./kg soil dw, respectively. The 56-d EC10, EC20 and EC50 based on reproduction were determined to be 1.17, 1.53 and 2.54 mg a.i./kg soil dw, respectively.
Reported statistics and error estimates:
The body weight change and reproduction data were tested for normal distribution and homogeneity of variance (α = 0.05) using the Shapiro-Wilk´s test and the Levene´s test, respectively. As the data for body weight changes and reproduction were normally distributed and homogeneous in both cases, Williams t-test was used to compare treatment and control values (multiple comparison, two-sided for weight and one-sided smaller for reproduction, α = 0.05). The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat® Solutions GmbH.

Table: Body weight changes of adult worms after 4 weeks

Treatment group

Body weight change per earthworm

mean ± SD*

mg

%

Control

115 ± 31

26.4 ± 8.1

1

114 ± 38

26.1 ± 9.6

2

98 ± 6

22.2 ± 2.6

4

133 ± 15

30.2 ± 4.5

8

124 ± 16

28.1 ± 4.0

16

128 ± 24

29.1 ± 6.5

* mean ± standard deviation of 4 replicates (8 in the control)

Table: Reproduction of the earthworms after 8 weeks

Treatment group

Number of juvenile earthworms

mean ± SD*

% of control

significance

Control

155 ± 29

-

-

1

168 ± 13

108.4

n.s.

2

155 ± 12

100

n.s.

4

140 ± 23

90.5

n.s.

8

164 ± 21

106.1

n.s.

16

158 ± 14

102.4

n.s.

* mean ± standard deviation of 4 replicates (8 in the control)

n.s. not significant

Validity criteria fulfilled:
yes
Remarks:
see 'Any other information on materials and methods incl. tables'

Description of key information

The (28- and) 56-d NOEC value is 16 mg/kg soil dw in earthworms (Eisenia fetida) based on reproduction, mortality and body weight.

Key value for chemical safety assessment

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

Additional information

The toxicity to soil macroorganisms was determined in a study according to OECD guideline No 222 and in compliance with GLP criteria (Ibacon GmbH, 2017). In this study, earthworms (Eisenia fetida) were exposed to nominal test substance concentrations of 0 (control), 1, 2, 4, 8 and 16 mg test item/kg soil dw for 28 days (4 weeks). The exposure time was 4 weeks for adult worms and 4 weeks for the offspring (an additional 28 days). The test substance was added to artificial soil that contained 10% peat, 69.65% quartz sand and 20% kaolin clay, by first adding the test item to quartz sand, followed by mixing with the soil. Test concentrations were not analytically verified and thus test concentrations are given as nominal. Mortality together with behavioural and morphological abnormalities of adult earthworms was determined at day 28 after application. Moreover, mean body weights were determined. Reproduction of the earthworms was determined by counting the number of juveniles after 56 days of application. No mortality was observed in any treatment group and no significant differences were found in body weights of adult worms. Moreover, no behavioural abnormalities were found and feeding activity of worms as comparable to the control. Also, reproduction rates were not statistically significant compared to the control. Based on these results, the 56 and 28-d NOEC values based on reproduction, mortality and growth (body weight) were determined to be 16 mg/kg soil dw.