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Toxicity to soil macroorganisms except arthropods

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Endpoint:
toxicity to soil macroorganisms except arthropods: short-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 Sep 1995 to 10 Oct 1995
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
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
yes
Remarks:
Acetone
Details on preparation and application of test substrate:
STOCK SOLUTION PREPARATION AND APPLICATION
Since the final net weight of the soil should be 750 g (final soil moisture content 40% of the dry weight which corresponds to 535.7 g dry soil weight) and the soil had an approximate original moisture content of 29.5% before the addition of the test substance, 693.7 g of artificial soil medium had to be added to each glass beaker. To obtain the correct final weight (750 g) and moisture content (40%), 56.3 mL of water and stock solution had to be added to the 693.7 g of soil. Since the test substance is not water soluble, the stock had to be prepared by diluting the test substance in acetone. During the first preliminary test, the appropriate stock was added to 10 g of sand. This treated sand was then added to each test vessel after the acetone had evaporated. However, it was observed that the test substance crystallised again on the sand and some of it remained on the vials used to prepare the treatments. Therefore, it was decided to perform a second preliminary and the definitive test, by adding the test substance solution directly onto the soil in order to ensure that the test substance was correctly dosed. The soil needed for all the replicates (plus some extra soil) was treated as a batch, by adding the stock solutions as the soil was being mixed with a Hebart Mixer (Model A 200 Hebart, Zurich, Switzerland). After the treatments were applied and the acetone had evaporated, the appropriate amount of soil (693.7 g) was added to each glass beaker. Thereafter, 56.3 g of deionized water was added to each beaker and the soil was mixed again with a mixing spindle connected to a drill. The control soil (batch of 3468.5 g) was treated with 75 mL of acetone. The test substance soil (batch of 3468.5 g) was treated with 75 mL of test substance in acetone stock solution. To obtain the needed test substance concentration of 1000 mg a.i./kg dry soil (corresponding to 535.7 mg a.i./535.7 g. of dry soil) a stock solution with a nominal concentration of 36.22 mg/mL (equivalent to 35.71 mg a.i./mL) was prepared by diluting 3622 mg of the test substance with acetone to a total volume of 100 mL. By adding 75 ml of this stock to the 3468.5 g of soil (dry weight 2716.5 g) we obtained a concentration of 1000 mg a.i./kg dry soil.
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: Earthworms
- Source: Gier-Angel, Neuenhof, Switzerland.
- Age at test initiation: All earthworms used in the study were at least 2 months old, mature organisms with clitellum.
- Weight at test initiation: Earthworms within a weight range of 300 to 600 mg were used for testing.

CULTURING
- Culturing conditions: The cultures were maintained in a medium consisting of peat and commercial garden soil and fed with horse manure. The ambient air temperature in the culture area was controlled in order to maintain the culture medium temperature at 20 ± 2 °C. Light intensity on the culture medium surface was approximately 624 lux.

ACCLIMATION
- Acclimation period: One day prior to test initiation, the earthworms needed for the test (and an additional 30%) were taken from the cultures and placed on artificial soil for acclimation.
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
yes
Total exposure duration:
14 d
Test temperature:
18.5 - 21.5 °C
pH:
- At test start: 5.58 (control); 5.65 (test substance treatment)
- At test end: 5.63 (control); 5.70 (test substance treatment)
Moisture:
- At test start: 35.59% (control); 34.80% (test substance treatment)
- At test end: 33.74% (control); 32.97% (test substance treatment)
Details on test conditions:
TEST SYSTEM
- Test container: 1.5 L glass beakers
- Amount of soil or substrate: Each replicate test vessel contained 750 g of the moist artificial soil (soil moisture content 40% of the dry weight* which is equivalent to 535.7 g of dry soil.
- Type: Closed; test vessels were covered with a glass lid to prevent excessive water evaporation, but allowing gas exchange.
- No. of organisms per container: 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 4
* Soil moisture content is 5% higher than the one recommended by the OECD Guideline 207. experience with earthworm culturing has shown that at a moisture content of approximately 40%, the earthworms develop better than at 35%.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition: The artificial soil medium consisted of 70% industrial sand (Zimmerli Mineralwerk AG, Zurich, Switzerland), 20% kaolin clay (Paut·Hauser & Company AG, Stafa, Switzerland) and 10% sphagnum peat moss (Jumbo, Arbon, Switzerland).
- Organic carbon (%): Not reported
- Maximum water holding capacity (in % dry weigth): Not reported
- CEC: Not reported
- Pretreatment of soil: CaCO3 was added in order to obtain a soil batch with a pH of 6.1. After adding deionized water the soil moisture was 29.5%. The soil was mixed with a cement mixer. The pH and the moisture content of the soil was re-measured at test initiation. The measured moisture content based on the dry soil weight after addition of the treatment solutions was 35.59% and 34.80% for the control and the 1000 mg a.i./kg treatment, respectively. The pH of the soil was 5.58 and 5.65 for the control and the 1000 mg a.i./kg treatment, respectively.
- Soil analysis: A sample of the artificial soil made up with the same batches of ingredients as the soil used during the definitive test were analysed by Lancaster Laboratories, Inc., Lancaster, PA, USA for the presence of pesticides, metals and PCBs. None of these compounds were detected at concentrations considered toxic in the soil sample analysed. No pesticide residue analysis was performed on the deionized water since its presence was not expected.

OTHER TEST CONDITIONS
- Photoperiod: Continuous
- Light intensity: 721 lux
- Soil quality measurements: pH and moisture content were measured from 2 soil samples each. The first sample was taken from replicates A and B and the second sample from replicates C and D from each treatment level. pH of the soil was measured on Day 0 and 14, using a Methrohm Model 692 pH meter (Methrohm AG, Buchs, Switzerland). Soil samples were removed from the beakers on Test Day 0 and 14, in order to determine percent moisture content orthe artificial soil. The samples were weighed and then dried at approximately 105°C. The dry weight of each sample was then determined and the percent moisture was calculated. Air temperature in the environmental chamber was continuously monitored using a Thermo-hygrometer from Fa. Lambrecht, Gottingen, Germany. Light intensity at the soil surface was measured using an IL 1400A light meter from International Light Inc., Newburyport, MA, USA.

EFFECT PARAMETERS MEASURED: Mortality and sub-lethal effects
Mortality and biological observations (e.g., colour changes and burrowing time) of earthworms were recorded on Day 0, 7 and 14 (test termination). To assess the condition of the earthworms on Day 7, replicate test vessels were individually emptied onto a tray where the earthworms were sorted from the soil and assessments were made (i.e. health and mortality). After performing the observations, the soil was returned to the appropriate vessel and the surviving earthworms were returned to the soil surface, where burrowing time was recorded again. At termination, the earthworms were sorted from the soil and health assessments were performed. These assessments generally included identification of healthy, missing, dead, and/or lethargic earthworms. The surviving earthworms from each test vessel were individually weighed after being rinsed with deionized water and blotted dry with paper towels. Earthworms were euthanized by freezing and later discarded.

VEHICLE CONTROL PERFORMED: yes

PRELIMINARY STUDY
Prior to initiation of the definitive test, 2 preliminary range-finding tests were conducted exposing earthworms to nominal concentrations ranging from 0.01 to 1000 mg a.i./kg soil dry weight, following a geometric progression of 10 and a control. At test termination of the first preliminary test (Day 14), mortalities of 0%, 0%, 0%, 10%, 20%, 0% and 10% were observed in the control, 0.01, 0.1, 1, 10, 100 and 1000 mg a.i./kg. treatment, respectively. At test termination of the second preliminary test (Day 14), mortalities of 0%, 0%, 0%, 0%, 10%, 10% and 0% were observed in the control, 0.01, 0.1, 1, 10, 100 and 1000 mg a.i./kg treatment, respectively. Based on these results, the definitive limit-test was performed at a nominal test substance concentration of 1000 mg a.i./kg dry soil weight and a deionized water control.
Nominal and measured concentrations:
- Nominal concentrations: 0 (control), 1000 mg/kg (limit concentration)
Reference substance (positive control):
no
Key result
Duration:
14 d
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Details on results:
- Burrowing time: On Test Day 0, no difference in burrowing time between the control and the test substance treated earthworms was observed. On Test Day 7, the earthworms exposed to the test substance had a significantly higher burrowing time (8.3 minutes) when compared to the earthworms exposed to the control (4 minutes). No conclusions on repellency regarding the test substance can be drawn from the results of these observations. An overview of the results on burrowing time is presented in 'Any other information on results incl. tables'.
- Mortality: Days 7 and 14 the average percent mortality for the control earthworms was 0%. The mean percent mortality in the 1000 mg a.i./kg treatment was 5% and 7.5% on Test Day 7 and Test Day 14, respectively. The difference between the control and the test substance treatment mortality was not significant. An overview of the results on mortality is presented in 'Any other information on results incl. tables'.
- Mean weight and weight difference: The weights of the earthworms exposed to the control remained stable over the 14 day exposure period, but the earthworms exposed to the test substance lost weight (63.8 mg). These results suggest that the test substance has an effect on earthworm metabolism. If the effect is direct (e.g. earthworms digesting the test material), or indirect (more movement of the earthworms due to the test material and therefore, higher loss of energy can, however not be determined with this type of test. The mean weights of earthworms alive on Test Day 0 and the weight difference between Test Day 0 and Test Day 14 are presented in 'Any other information on results incl. tables'.
Reported statistics and error estimates:
Mortality data derived from the acute test was insufficient to estimate a median lethal concentration (LC50), since mortality in the test substance treatment was < 50%. The effects of treatment with the test substance on earthworm mortality was tested for significance against the control using the Fisher's Exact test (Zar, 1984). The effects of treatment with the test substance on earthworm mean body weight and burrowing time were tested for significance against the control data using the Mann-Whitney U test (Zar, 1984) because the assumption of normality was not met (Shapiro-Wilk's test; Weber et al., 1989). The computer program used to run the statistical analysis was "Statistica for Windows", by StatSoft Inc., Tulsa OK, USA.

Table: Burrowing time of earthworms (Eisenia foetlda) on Test Day 0 and 7, of the 14-day acute toxicity test with the test substance

Treatment

Mean Burrowing Time(1) ± SD (in minutes)

Day 0

Day 7

Control

4.5 ± 1.0

4.0 ± 0.8

1000 mg a.i./kg test substance

4.3 ± 0.5

8.3 ± 1.5*

1) Burrowing time is the time needed for the ten earthworms to burrow completely in the soil after they.were initially placed on the soil surface.

* Significant difference between the control and the test substance treatment level (p0.05, Mann-Whitney U test).

 

Table: Percent mortality of earthworms (Eisenia foetida) during the 14-day acute toxicity test with the test substance

Treatment

Percent Mortality

Day 7

Day 14

Replicates

A

B

C

D

Mean ± SD

A

B

C

D

Mean ± SD

Control

0

0

0

0

0 ± 0

0

0

0

0

0 ± 0

1000 mg a.i/kg test substance

0

10

0

10

5 ± 5.8

0

20

0

10

7.5 ± 9.6

Note: No significant difference between the test substance treatment and the control (p > 0.05, Fisher's Exact test).

 

Table: Mean weights of earthworms (Eisenia foetida) determined at test initiation and mean weight difference of surviving earthworms at test termination of the 14-day acute toxicity test with the test substance

Treatment

Mean Earthworm Weight (mg) ± SD

Day 0(1)

Weight Difference(2) Day 0-Day 14

Control

349.3 ± 50.7

-11.9 ± 13.2(3)

1000 mg a.i./kg test substance

342.3 ± 38.7

63.8 ± 19.2*

* Significant difference between the control and the test substance treatment levels (p ≤ 0.05, Mann-Whitney U test).

1) No significant difference between the control and the test substance treatment levels (p >0.05, Mann-Whitney U test).

2) Weight difference= weight Test Day 0 - weight Test Day 14 (positive values indicate a weight loss, while negative values indicate a weight gain).

3) Earthworms weight gain in the control during the 14 day exposure period not

significant (p> 0.05, Mann-Whitney U test)

Validity criteria fulfilled:
yes
Conclusions:
Based on the results of this study, the 14-Day LC50 value for. the earthworms exposed to the test substance was empirically estimated to be > 1000 mg a.i./kg soil dry weight. The NOEC could not be determined, since significant effects of test substance treatment on burrowing time and weight loss could be determined during the limit-test at a concentration of 1000 mg a.i./kg soil dry weight.
Executive summary:

The toxicity to soil macroorganisms was determined in a GLP-compliant study according to OECD guideline No 207. In this study, earthworms (40 per treatment; Eisenia fetida) were exposed to nominal test substance limit concentration of 1000 mg test substance/kg soil dry weight along with a deionized water control for 14 days. The test substance was added to artificial soil that contained 70% industrial sand, 20% koalin clay and 10% sphagnum peat moss. The artificial soil had a pH of 5.58 - 5.65 and a percent moisture content of 34.80 - 35.59% at study initiation. Test concentrations were not analytically verified and thus test concentrations are given as nominal. Burrowing time was determined at Day 0 and 7 and mortality at Day 7 and 14. Furthermore, mean weights of earthworms were determined at test initiation and termination and a mean weight difference was calculated. At Day 7, the earthworms exposed to the test substance had a significantly higher burrowing than the earthworms exposed in the control. No mortality was observed in the control group throughout the study. Although mortality was observed in the 1000 mg test substance/kg soil dw was observed (7.5% at Day 14), it was not considered significant relative to the control. The weights of the earthworms exposed to the control remained stable over the 14 day exposure period, but the earthworms exposed to the test substance lost weight. Based on these findings, the 14 -d LC50 was determined to be > 1000 mg test substance/kg soil dw.

Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 Jul 1997 to 17 Sep 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: BBA-Richtlinie VI, 2-2, (with respect to Draft ISO/ DIS 11268-2, 1993)
Version / remarks:
1994
GLP compliance:
yes
Analytical monitoring:
yes
Vehicle:
no
Details on preparation and application of test substrate:
The following stock solutions were prepared:
- Test substance, application of one-fold field rate: 54.6 mg of test substance was mixed with and made up to 40 mL with deionised water.
- Test substance, application of five-fold field rate: 270.8 mg of test substance was mixed with and made up to 40 mL with deionised water.
- Reference substance, application corresponding to 1000 g Benomyl / ha: 133.0 mg reference substance were mixed with and made up to 40 mL with deionised water.
These stock solutions were each mixed ultrasonically for 15 minutes. 1.35 mL of the stock solutions were then sprayed on the 198 cm2 soil surface of the corresponding test vessels using laboratory spraying equipment (simulation of field application).
- Loss amount: The amount of loss (amount not sprayed on soil surface) was in the range of 0 - 3 % for the test and reference substance.
- Preparation control: The water controls were treated accordingly using deionised water only for spraying. The amount of loss measured in one test container was 4%.
- Evaporation of vehicle before use: After the application, the containers were left open for 0.5 - 1 hour under artificial light to enable a possible solvent evaporation of the test substance dilution. Thereafter, containers were closed and transfered to the exposure chamber (start of exposure).
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: Earthworms
- Source: Jean-Marc Moulin, SARL Moulin, 7 rue du Pont de Bacqueville, F-60510, Litz, France
- Age at test initiation: ≥3 months, adult, with clitellum
- Weight at test initiation: Average of 443 mg (390-490 mg) based on mean weights of 10 earthworms per test vessel in the control and the application rates of the test substance, 425 mg (390-450 mg) in the test vessels of the reference substance.

CULTURING
- Handling: Care was taken to minimise possible stress due to handling. Worms which were damaged or dropped during the transfer were not used for the exposure.
- Culturing conditions: Earthworms were held in a mixture of approximately 50% peat and 50% cattle manure for 9 days prior to testing.
- Treatment: Earthworms were not treated against a disease during holding.
- Feeding: The earthworms were fed with commercially available dried cattle manure, until 24 hours prior to the test.

ACCLIMATION
- Acclimation period: 24 hours conditioning in soil used for the test.
- Acclimation conditions: Same as test
- Health during acclimation (any mortality observed):
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:
20.5°C
pH:
- At test start: 6.4 - 6.5
- At test end: 6.7
Moisture:
- At test start: 62 - 66% (Moisture content of artificial soil related to Water Holding Capacity)
- At test end: 49 - 57% (Moisture content of artificial soil related to Water Holding Capacity)
Details on test conditions:
TEST SYSTEM
- Test container: Closed polystyrol containers of 1 L (18.3 x 13.6 x 6 cm; 198 cm2 surface area on top of soil) with plastic covers (with a central hole covered with fine mesh netting)
- Amount of soil or substrate: 750 g wet weight of soil corresponding to 428 g dry weight of soil
- No. of organisms per container: 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 4
- Food: Dried cow manure of a quality shown to be capable of maintaining the earthworm population was fed. One day before the exposure of the organisms, 20 g finely ground cattle manure per kg artificial soil were added as food base for the worms. During the first 28 days ( 4 weeks), 5 g of cattle manure were scattered on the surface of each container one day after the application of the test substance and weekly thereafter (days 7, 14, 21 and 28), except for the reference substance, where feeding was stopped after 7 days since the food consumption was very low. The food was moistened by the addition of 5 ml of drinking (tap) water. No feeding was done during the last 28 days (4 weeks) after adult worms had been removed.
- Introduction of individuals: After the preparation of the artificial soil and distribution in the test containers, the washed, dried (filter paper) and weighed test organisms were randomly placed on the surface of the artificial soil and allowed to enter the soil. At least half an hour later and after all the worms had buried, the test substance was sprayed on the surface of the artificial soil with an amount of water corresponding to 600 L/ha.

SOURCE AND PROPERTIES OF SUBSTRATE
- Composition: 10% Sphagnum peat (% related to dry weight), 20% Kaolinite clay, 68-69% Industrial quartz sand and approximately 1% calcium carbonate to reach a pH of 6±0.5
- Storage (condition, duration):

OTHER TEST CONDITIONS
- Photoperiod: light/ dark cycle of 16 h / 8 h was maintained throughout the period of the test.
- Light intensity: 550 - 800 lux

SOIL QUALITY MEASUREMENTS
The water holding capacity was determined prior to the start of the test by comparing the weight of water saturated soil (and subsequent draining) with that of soil dried to constant weight. The pH was measured at the start of the test in each application rate with a Methrom pH-meter 632 using a combined glass electrode of Orion Ross Sure-Flow. In addition, the moisture content was determined at the start and the end of the exposure by drying the samples for 25 h in a Heraeus drying oven. The temperature (°C) during exposure in the climatic chamber Type KBP 6395 F U was recorded continuously.

EFFECT PARAMETERS MEASURED: Adult mortality, adult sub-lethal effects and reproduction
After 4 weeks, adult worms were removed from the soil and observations of mortality and sub lethal symptoms, such as biomass, flaccidity and abnormal behavioural activity were made. After another 4 weeks, the young worms were extracted from the soil by placing the containers in a waterbath at around 70°C. All emerging were removed and counted. In addition, the soil of each container was distributed on a tray and checked by hand for additional worms that may have remained in the soil. The number of hatched worms per vessel were determined.
Nominal and measured concentrations:
- Nominal application rates: 0 g/ha (deionised water control), 931 g test substance/ha (corresponding to one-fold field rate of 200 g a.i./ha) and 4616 g test substance/ha (corresponding to five-fold field rate of 1000 g a.i./ha).
Reference substance (positive control):
yes
Remarks:
1000 g Benomyl / ha or to effectively applied 1134 g Benomyl / ha.
Key result
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
4 616 g/ha
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
1.54 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: recalculation to mg/kg soil dry weight unit by the registrant
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
4 616 g/ha
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
4 616 g/ha
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: biomass
Details on results:
- Mortality: In the control and the application rates 931 and 4616 g test substance/ ha, mortality was 0%.
- Feeding behaviour: During the 28 days exposure of adults, the earthworms of the control and the test substance were fed a total of 25 g finely ground cattle manure by scattering on the soil surface. This food was added in weekly portions of 5 g and the portion was eaten within 7 days.
- Biomass: The average live weight of the earthworms exposed to 931 and 4616 g test substance/ ha increased by 30 and 36% of the initial weight, respectively. The average live weight of the earthworms in the control recorded at the end of the exposure was 30%.
- Offspring production: The mean number of offspring in the application rates 931 and 4616 g test substance/ ha was 80 and 83, respectively, whereas 74 were counted in the control.
An overview ofthe results is presented in 'Any other information on materials and methods incl. tables'.
Results with reference substance (positive control):
- Mortality: Mortality in the reference stubstance was 0%.
- Feeding behaviour: The earthworms of the reference substance received a total of 10 g since the food consumption was very low.
- Biomass: The average live weight increase of the earthworms recorded in the reference substance treatment was significantly lower compared to the increase in the control (18% vs 30%).
- Offspring production: With the application of 1134 g / ha Benomyl, the mean number of offspring was 3. This was a statistically significant reduction compared to the control.
Reported statistics and error estimates:
Results of the toxicity test were interpreted by standard statistical techniques using SAS PROC GLM and PROC NLIN (SAS 1990a) of SAS Version 6.11 (SAS 1990b). All tests were performed using a significance level of α= 0.05. No statistical analysis of the measured data, such as biomass, were performed, since no reduction, but an increase of the data was observed. Dunnett's multiple means comparison test (Dunnett, 1955 & 1980) using mean values per replicate as experimental units was run. No statistical analysis for mortalityor abnormality of the data were performed, since no such effects occured.

Table: Mass of live adult worms at the start and after 4 weeks of exposure

Application rate

Average live weight (mg)

Start exposure

End exposure

% of initial weight

Per vessel

Per conc.

Per vessel

Per conc.

Per vessel

Per conc.

Blank

450

445

620

580

38

30

450

560

24

430

540

26

430

600

33

 

 

 

931 g/ha test substance

390

433

490

563

26

30

460

600

30

400

540

35

480

620

29

 

 

 

4616 g/ha test substance

470

453

630

615

34

36

420

530

26

490

680

39

430

620

44

 

 

 

Mean

 

443

 

586

 

32

Min

 

433

 

563

 

30

Max

 

453

 

615

 

36

1134 g/ha reference substance

420

425

460

500

10

18*

390

480

23

450

570

27

440

490

11

 

 

 

* Statistically significant difference compared to control according to a Dunnett-test (P≤0.05)

 

Table: Number of offspring per test container at the end of the test

Application rate

Number of offspring per test container at the end of the test

Mean offspring

Standard Deviation

Coefficient of Variation of Means (%)

P-value of Trend test

1

2

3

4

Blank

91

52

80

71

74

16.5

22

 

931 g/ha test substance

77

65

79

99

80

14.1

18

0.8588

4616 g/ha test substance

67

91

106

69

83

18.7

22

0.6643

1134 g/ha reference substance

7

2

1

3

3

2.6

81

0.0001*

* Statistically significant difference compared to water (blank) control

Validity criteria fulfilled:
yes
Remarks:
See 'Any other information on materials and methods incl. tables'
Conclusions:
The NOECs (28 d / 4 weeks) of the test substance in regard to mortality and biomass of adults, as well as in regard to the number of offspring after an exposure of 56 days (8 weeks) was 4616 g test item / ha based on the effectively applied application rate; this amount corresponds to 1.54 mg active ingredient/kg soil dw. Therefore, the test substance is "non-toxic to earthworms" when applied at the application rates tested, based on the inherent toxicity of the test substance in this chronic test.
Executive summary:

The toxicity to soil macroorganisms was determined in a GLP-compliant study according to BBA Guideline part VI, 2-2. In this study, earthworms (40 per treatment; Eisenia fetida) were exposed to nominal test substance application rates of 0 (control), 913 and 4616 g/ha test substance/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, 68 - 69% 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 sublethal effects 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 4616 g test item/ha soil dw, which corresponds to 1.54 mg active ingredient/kg soil dw.

Description of key information

All available data were assessed and the studies representing the worst-case effects were included as key or weight-of-evidence studies. Other studies are included as supporting information. The key studies are considered to be worst-case and were selected for the CSA.

The 14-d LC50 value is >1000 mg/kg soil dw in earthworms (Eisenia fetida), OECD TG 207, Candolfi 1995. The 56 (28-)d NOEC value is 4616 g/ha soil dw in earthworms (Eisenia fetida), BBA Part VI, 2-2, Rufli 1997.

No quantitative acute effect value (LC50) can be determined as no ≥50% effect was reached in the study.

Key value for chemical safety assessment

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

Additional information

Table: Overview of available data on the toxicity to macroorganisms

Soil type

Guideline / GLP

Endpoint

Effect value

Comment

Reference

Artificial soil (70% industrial sand, 20% kaolin clay and 10% sphagnum peat moss)

OECD 207

14-d LC50

>1000 mg/kg soil dw

Limit test

Candolfi, 1995

Artificial soil (68-69% industrial sand, 20% kaolin clay, 10% sphagnum peat and ~1% calcium carbonate)

BBA Part VI, 2-2

56 (28-)d NOEC

4616 g/ha soil dw

(=1.54 mg/kg soil dw; recalculated by the registrant)

The NOEC is based on reproduction, mortality and body weight.

Due to the experimental design only having two concentrations of the test item, no meaningful statistical analysis could be conducted and no reliable EC10 or EC20 values were

calculated.

Rufli, 1997