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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:
2015-07-16 to 2015-09-16 (exposure period)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Name of test material (as cited in study report): 2-methylaminoethanol
- Physical state: liquid / yellowish and clear, homogeneous
- Expiration date of the lot/batch: 11 November 2016
- Storage condition of test material: Storage at room temperature under nitrogen
- log Pow: -0.91
- Water solubility: miscible
- Test substance no.: 07/0540-3
- Date of production: 11 November 2014
Analytical monitoring:
no
Vehicle:
no
Details on preparation and application of test substrate:
- Method of mixing into soil (if used): The water solubility is enough to prepare a sufficient stock solution with deionized water.
Application of the test substance: dissolved in deionized water. Two test substance stock solution were prepared:
Stock solution 1: Initial weight: 12500.8 mg/L
Stock solution 2: Initial weight: 1250.2 mg/L
The amounts of the test substance were weighed into 1000 mL volumetric flasks. Both test substances were colourless and clear. The test substance was completely dissolved, verified by visual inspection. The pH values of both stock solutions were corrected in a range 6.0-7.0. The required amounts of artificial soil were weighed into a mixing bowl. The aliquots of the test- or reference substance stock solution were dosed to a measuring cylinder. Required volumes of deionized water were also added to reach a total volume of 630 mL. By addition of this volume of aqueous solution, the content of water of the test substrate was adjusted to about 50% WHCmax. After mixing the solutions were added to the artificial soil in the mixing bowl. After thoroughly mixing with an electric mixer, the test substrates were distributed to the test containers. The test units of control were prepared only with deionized water, without test substance.
- Controls: control (due to the high number of replicates the test assay of control was prepared in two portions)
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: earthworm
- Source: Agricultural Center Limburgerhof of BASF SE, D-67056 Ludwigshafen, Germany
- Age at test initiation (mean and range, SD): ca. 7 months (with clitellum)
- Weight at test initiation (mean and range, SD): 250 - 600 mg

ACCLIMATION
- Acclimation period: overnight
- Acclimation conditions (same as test or not): yes (acclimatised in artificial soil; 50 ± 10 % of the max. water holding capacity [WHCmax])
The worms selected for the test, were acclimatized in the test substrateunder test conditions overnight under permanent light without feeding before starting the test.
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
56 d
Remarks:
survived adult worms removed from substrate and counted after 28 d; juvenile worms counted after 56 days
Test temperature:
21.0-21.6°C
pH:
5.8 - 6.2
Moisture:
50 ± 10 % WHCmax
Details on test conditions:
TEST SYSTEM
- Test container (material, size): Plastic dish with a volume of about 1.0 L, dimensions 11*15.5*6 [cm] with transparent and punctured lid
- Amount of soil or substrate (per test container): 694 g artificial soil (wet weight)
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8
- No. of replicates per reference: 4

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate): artificial soil according to OECD 222
- 10.6 % sphagnum peat (finely ground)
- 20.9 % kaolin clay
- 68.2 % quartz sand
- 0.3 % calcium carbonate (CaCO3)
- water holding capacity: 52.2g/100 g Dw (mean of 2 single values 51.9 g/52.6 g)
- Storage (condition, duration): not reported

OTHER TEST CONDITIONS
- Photoperiod: light/dark : 16 h/8 h
- Light intensity: 624 Lux, (mean of 5 single measurements: 742, 602, 622, 643 and 671 [Lux])

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
-mortality/biomass: After 28 days of exposure the worm mortality was assessed by emptying the containers onto suitable trays, sorting the adult worms from the test substrate and - if necessary - testing their reaction to a mechanical stimulus. Additionally, the live weight of the adult worms was determined.
- reproduction: After 56 days juvenile worms were sampled out of the test containers. Extraction was done using the water bath method (temperature of the water bath approx. 60° C). During this process morphological and behavioral changes and the number of juveniles in each test container were recorded.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study: not performed
Nominal and measured concentrations:
nominal: control, 62.5, 125, 250, 500 and 1000 mg/kg artificial soil (dw)
Reference substance (positive control):
yes
Remarks:
Carbendazim (BAS 346 F), CAS# 10605-21-7
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: adult earthworms
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: body weight change
Remarks on result:
other: adult earthworms
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
250 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Duration:
56 d
Dose descriptor:
EC10
Effect conc.:
201 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Remarks on result:
other: LCL: 158 UCL: 256 mg/kg soil dw
Duration:
56 d
Dose descriptor:
EC50
Effect conc.:
637 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Remarks on result:
other: LCL: 569 UCL: 713 mg/kg soil dw
Details on results:
- Mortality at end of exposure period (28 d): 0 % (all replicates)
- Changes in body weigth of live adults (% of initial weight) at end of exposure period: control: +51.66 %
- No. of offspring produced: control: 267 (mean of 8 replicates)
- Morphological abnormalities: no
- Behavioural abnormalities: no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: number of juveniles (control) = 267 +/- 18.06, n = 8
number of juveniles (ref. substance) = 26.5 +/- 8.74, n = 4, inhibition = 90.07 %
Wilcoxon test (one-sided -): p<=0.01
Reported statistics and error estimates:
Calculations to determine the NOEC/LOEC and the ECx calculations were carried using SAS System computer software.

For changes in body weight of live adults (% of initial weight) after first four weeks the statistical evaluation was carried out Williams trend test. If this test is not significant the Dunnett’s test for a simultaneous comparison of several dose groups with the control was carried out. The statistical unit was the test container. The tests were performed onesided. For the number of juveniles (offsprings) the Jonckheere-Terpstra trend test was performed. If this test is not significant, a pair-wise comparison of each dose group with the control was carried out using Wilcoxon-test. The statistical unit was the test container.
The tests were performed one-sided.

In addition, an ECx calculation was performed for the changes in body weight of live adults and the offsprings. The percent reduction was calculated in relation to the control mean. Percentages lower than zero were set to zero. These values were used for ECx calculations with the probit model. The 95 % confidence intervals were determined via normal approximation.

For the reference substance, an analysis of the number of juveniles (offsprings) a comparison with the control was performed using the Wilcoxon test. The statistical unit was the test container. The tests were performed one-sided.

Biomass change of the earthworms after 28 d of exposure and number of offspring after 56 d:

Concentration

[mg test

substance /kg

soil(dw)]

Offspring (after 56 d)

Body weight change (after 28 d)

Number of Offspring

(Mean ± SD)

Related to

Control [%]

Mean

[%]

Related to

Control [%]

Control

267.00 ± 18.1

-

51.66

-

62.5

282.75 ± 22.3

105.90

44.58

-13.70

125

272.75 ± 29.3

102.15

56.51

9.39

250

222.00 ± 27.2

83.15

51.58

-0.15

500

168.00 ± 26.1

62.92

46.98

-9.06

1000

79.00 ± 7.1

29.59

42.49

-17.75

Summary of pH values at start and end of exposure:

Nominal test

concentration

[mg/kg Dw]

 

pH

(at test initiation)

pH

(at test end)

0 (control)

5.9

6.2

62.5

5.8

6.2

125

5.8

6.2

250

5.9

6.2

500

5.9

6.2

1000

5.9

6.2

(Samples were taken from replicate 1 of each test concentration)

Validity Criteria

Mean mortality in the control should be ≤ 10 %. The reproduction in the controls should be at least 30 juveniles per container and the coefficient of variance of reproduction in the control should be ≤ 30 %.

All validity criteria for the study were met. The mean mortality in control and solvent control was 0 %, the mean value for reproduction in the control was 267 +/- 18.1 juveniles per container. The coefficient of variance of reproduction in control was 6.8 %. No deviations from the test guideline or other incidents occurred during the course of the reported test which may have influenced the results.

Validity criteria fulfilled:
yes
Executive summary:

In a 56 day study adult earth worms of the species Eisenia fetida were exposed to 2-methylaminoethanol at nominal concentrations of 0 mg (control), 62.5 mg, 125 mg, 250 mg, 500 mg and 1000 mg/kg dry weight in accordance with the OECD 222 guideline. The purpose of this study was to determine a NOEC/LOEC and/or an EC50 for the effects of 2-methylaminoethanol on the reproduction (56 days after application), mortality and the biomass development (28 days after application) of the earthworm Eisenia fetida by dermal and alimentary uptake using a standardized artificial soil.

The mortality and the body weight change of the adult worms was observed and recorded on day 28.

The reproduction, mean number of juveniles per test concentration 56 days after application was observed.

Test results: Adult worms

No mortality of adult worms was observed in the controls and at all replicates with test substance

NOECMortality: ≥ 1000 mg test substance/kg soil dw

LOECMortality: > 1000 mg test substance /kg soil dw

Body weight change

NOECBiomass change: ≥ 1000 mg test substance/kg soil (dw)

LOECBiomass change: > 1000 mg test substance /kg soil (dw)

Reproduction by count of juvenile worms:

NOECReproduction: = 250 mg test substance/kg soil dw

LOECReproduction: = 500 mg *test substance/kg soil dw

ECx values [mg/kg soil dw], conf. limits 95 %

EC10: 201 LCL: 158 UCL: 256

EC50: 637 LCL: 569 UCL: 713

The results in this study are consistent with all validity criteria and the test is valid according to the test guideline of this study. No deviations from the test guidelines or other incidents occurred during the course of the reported test, which may have influenced the results.

Description of key information

In a 56 day study adult earth worms of the species Eisenia fetida were exposed to 2-methylaminoethanol at nominal concentrations of 0 mg (control), 62.5 mg, 125 mg, 250 mg, 500 mg and 1000 mg/kg dry weight in accordance with the OECD 222 guideline (BASF, 2016).

The mortality and the body weight change of the adult worms was observed and recorded on day 28.

The reproduction, mean number of juveniles per test concentration 56 days after application was observed.

Test results: Adult worms

No mortality of adult worms was observed in the controls and at all replicates with test substance

NOECMortality: ≥ 1000 mg test substance/kg soil (dw)

LOECMortality: > 1000 mg test substance /kg soil (dw)

Body weight change

NOECBiomass change: ≥ 1000 mg test substance/kg soil (dw)

LOECBiomass change: > 1000 mg test substance /kg soil (dw)

Reproduction by count of juvenile worms:

NOECReproduction: = 250 mg test substance/kg soil (dw)

LOECReproduction: = 500 mg *test substance/kg soil (dw)

ECx values [mg/kg soil(dw)], conf. limits 95 %

EC10: 201 LCL: 158 UCL: 256

EC50: 637 LCL: 569 UCL: 713

The results in this study are consistent with all validity criteria and the test is valid according to the test guideline of this study. No deviations from the test guidelines or other incidents occurred during the course of the reported test, which may have influenced the results.

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:
637 mg/kg soil dw
Long-term EC10, LC10 or NOEC for soil macroorganisms:
201 mg/kg soil dw

Additional information