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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:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
from 1994-08-25 to 1994-09-08
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
EU Method C.8 (Toxicity for Earthworms: Artificial Soil Test)
Principles of method if other than guideline:
Toxizitaetstest fuer Regenwuermer nach 88/302 EWG
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on preparation and application of test substrate:
- Method of mixing into soil: The test substance was mixed with silica sand and afterwards added to soil substrate in a plastic bag. Water was added to a theoretical water content of approx. 40% and the substrate was thoroughly mixed
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: earthworm
- Source: Eisenia GmbH&Co. Produktions- und Vertrieb KG, Kapellenstraße 25, Wiesbaden, Germany
- Age at test initiation: at least 2 months
- Weight at test initiation: 307-594 mg

ACCLIMATION
- Acclimation period: not mentioned
Study type:
laboratory study
Substrate type:
other: mixture of peat and horse dung (1:1)
Limit test:
yes
Total exposure duration:
14 d
Post exposure observation period:
no
Test temperature:
20 ± 2 °C
pH:
5.5
Moisture:
37 - 39 %
Details on test conditions:
TEST SYSTEM
- Test container (material, size): Makrolon cages with perforated plexiglass covers, size not mentioned
- Amount of soil or substrate: not mentioned
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 4
- No. of replicates per vehicle control: not applicable

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate): mixture of peat and horse dung (1:1)
- Organic carbon (%): not mentioned
- Maximum water holding capacity (in % dry weight): not mentioned
- CEC: not mentioned
- Pretreatment of soil: not mentioned
- Storage (condition, duration): fresh prepared
- Stability and homogeneity of test material in the medium: not mentioned

OTHER TEST CONDITIONS
- Photoperiod: 24 h
- Light intensity: approx. 400-600 lux

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable, limit test
- Range finding study: not performed
Nominal and measured concentrations:
nominal concentration, not measured: 1000 mg/kg dry soil
Reference substance (positive control):
yes
Remarks:
chloracetamide, historical data
Duration:
14 d
Dose descriptor:
LC10
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Mortality at end of exposure period: 0/40 (0%) after 7 days; 1/40 (2.5%) after 14 days
- Total mass of adults at beginning of test: 406 mg (mean)
- Changes in body weight of live adults (% of initial weight) at end of exposure period: not mentioned
- No. of offspring produced: not mentioned
- No. of unhatched cocoons: not mentioned
- Morphological abnormalities: not mentioned
- Behavioural abnormalities: not mentioned
- Other biological observations: not mentioned
Results with reference substance (positive control):
RESULTS: TEST WITH REFERENCE SUBSTANCE (historical data, 1994-03-03 until 1994-03-17)
- Concentrations: 0, 10, 30 and 100 mg 2-chloroacetamide/kg dry soil
- Results (mortality): 10 mg/kg: 0/20 (0%) after 14 days; 30 mg/kg: 18/20 (90%) after 7 and 14 days, respectively; 100 mg/kg: 17/20 (85%) after 7 and 14 days, respectively.

Reported statistics and error estimates:
no statistics performed

RESULTS: EXPOSED

- Nominal concentrations: 1000 mg/kg dry soil

- Effect data (Mortality): 0/40 (0%) after 7 days; 1/40 (2.5%) after 14 days

RESULTS: CONTROL

- Effect data (Mortality): 0/40 (0%) after 7 and 14 days

Validity criteria fulfilled:
yes
Conclusions:
The structurally related substance 1,2-Benzenedicarboxylic acid, di-C6-10-alkyl esters was not toxic under test conditions at a limit concentration of 1000 mg/kg dry soil.
Executive summary:

The acute toxicity of the structurally related substance 1,2-Benzenedicarboxylic acid, di-C6-10-alkyl esters (WITAMOL 110) to the earthworm eisenia foetida was determined during a 14 day exposure period according to EEC Methods for Determination of Ecotoxicity, Directive 88/302/EEC. For the limit test with a concentration of 1000 mg/kg dry soil, the test substance was mixed with silica sand and afterwards added to soil substrate. After 7 and 14 days the effects of the test substance on the earthworms' survival were examined.

No mortality occurred at the tested concentration after 7 and 14 days. The 14 day LC50 of WITAMOL 110 to Eisenia foetida was observed to be > 1000 mg/kg dry soil.

Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Type of information:
experimental study
Adequacy of study:
key study
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))
Deviations:
yes
Remarks:
Food was provided on day 0 (test start) instead of day 1 due to the good experience with this procedure.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Tightly closed, at a dry and good ventilated place
- Solubility and stability of the test substance in the solvent/vehicle: Not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None.



Analytical monitoring:
yes
Details on sampling:
The test item was analysed in the treated soil subsequently to its preparation on day 0 and on day 55.
Vehicle:
no
Details on preparation and application of test substrate:
- Method of mixing into soil : The respective test item amount for each concentration was weighed out, dissolved in demineralised water and added to the artificial soil. Additional demineralised water was added to adjust the humidity of the artificial soil to a moisture of 40 – 60 % of the WHCmax. Subsequently, the test medium was mixed thoroughly to ensure a homogenous distribution and a soil amount corresponding to about 600 g soil DW will be filled into the test vessels.
- Controls: Untreated artificial soil
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: Earthworms
- Source: In-house culture
- Age at test initiation: 2-12 months (difference of age did not deviate by more than 1 month)
- Weight at test initiation (range): 0.40 to 0.54 g
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
56 d
Test temperature:
18 - 23°C (The room temperature increased to 23°C for about 7 hours. This deviation is considered to have no impact on quality and integrity of the study.)
pH:
5.7 - 6.1
Moisture:
24.3 - 29.7% (The soil moisture deviated by more than 10 % from the initial value. This deviation is considered to have no impact on quality and integrity of the study.)
Details on test conditions:
TEST SYSTEM
- Test container (material, size): Round plastic boxes with an inner diameter of 15.0 cm, corresponding to a bottom surface area of 177 cm2 and a height of 14 cm.
- Amount of soil or substrate: 610 g per test container
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8
- No. of replicates per vehicle control: Not applicable

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- The artificial soil consisted of the following components: 10 % peat, air-dried and finely ground, 20 % kaolin, kaolinite content > 30 %, 69 % air-dried quartz sand (sand with > 50 % particle size of 0.05 - 0.2 mm), 0.38 % calcium carbonate (CaCO3) to achieve a pH of 6.0.
- Pretreatment of the soil: Two days before experimental starting, the artificial soil (22 kg) was pre-moistened by adding 2442 g demineralised water to achieve a moisture content corresponding to 27 % of the maximum water capacity of the artificial soil. The moistened soil was kept covered to prevent evaporative water losses prior to the start of exposure.

OTHER TEST CONDITIONS
- Photoperiod: 16h
- Light intensity: 571 +/-93 lx

EFFECT PARAMETERS MEASURED:
A) no obvious pathological symptoms
B) no reaction to touch
C) no negative phototactical reaction
D) spontaneous segmentation and separation
E) spasmodic winding
F) yellow excretion from the oral aperture
G) ulcer and skin bleedings
H) dead earthworms

VEHICLE CONTROL PERFORMED: No

TEST CONCENTRATIONS
- Nominal test concentrations: 20 – 62.5 – 125 – 250 – 500 – 1000 mg/kg SDW
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study
- Test concentrations: 1 – 10 – 100 – 1000 mg/kg SDW
- Results used to determine the conditions for the definitive study: 10% mortality at 1000 mg test item/kg soil dry weight, no other effects observed.
Nominal and measured concentrations:
20, 62.5, 125, 250, 500, 1000 mg/kg SDW (nominal)
20.2-23.7, 64.1 - 91.5,
Reference substance (positive control):
yes
Key result
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:
mortality
Duration:
56 d
Dose descriptor:
LOEC
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
500 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
56 d
Dose descriptor:
LOEC
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:
EC10
Effect conc.:
504 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
56 d
Dose descriptor:
other: EC20
Effect conc.:
819 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
- Changes in body weight of live adults (% of initial weight) at end of exposure period: No significant differences in bodyweight compared to control.
- No. of offspring produced: The mean number of juveniles in the control was 118. The average number of juveniles in the treated groups ranged from 88 to 126
- No. of unhatched cocoons: None
- Morphological abnormalities: None
- Behavioural abnormalities: None
- Other biological observations: None
- Mortality at end of exposure period: None

Results with reference substance (positive control):
- Results with reference substance valid?
- Relevant effect levels: Significant effects of Carbendazim were observed between 1 and 5 mg a.s./kg soil DW, the validity criteria was fulfilled.

Measured Concentrations and Percent of the Nominal Concentrations of the Test Item LINPLAST 810 P during the Definitive Test at the Start of the Exposure (day 0)

Sampling date/Start of analysis  2016-12-14   2016-12-14  2016-12-14
Nominal concentration C8C8 component  C8C10 component    C10C10 component
 [mg/kg SDW]  

Meas. conc. [mg/kg SDW]

 %  

Meas. conc. [mg/kg SDW]

 %  

Meas. conc. [mg/kg SDW]

 %

 1000

1152

115

1317

132  

1283

 128

 500

577

115

 500

100

467

 93

 250

262

105

 259

 104

 207

 83

 125

135

108

 134

107

 97

 78

 62.5

64

103

71

114

92

146 

 20.0

20

101

20

102

24

118 

 Control

< LOQ

< LOQ

< LOQ

  

      

Reproduction Rate (Number of Juveniles after 56 days)

 Application rate [mg/kg SDW]  Mean ± SD    CV [%] [%] of control    Sig.
Control  118 ± 23.9  20.3  -
20.0  107 ± 15.3 14.3   90.7     No

62.5

126 ± 10.3

 8.17  

   107

 No

125

 107 ± 7.68

 7.18

 90.7

No 

250

 122 ± 17.8

 14.6

 103

 No

500

 99 ± 16.0

16.1 

 83.9

 No

1000

 88 ± 15.3

 17.3

 74.6

 Yes

Validity criteria fulfilled:
yes
Conclusions:
No mortalities, significant pathological symptoms or changes in the behaviour of adult earthworms were observed in the control or any test item treatments. The reproduction was significantly effected at 1000 mg/kg SDW and therefore the lowest NOEC of the test item based on reproduction was determined to be 500 mg/kg SDW. The corresponding EC10- and EC20-values for reproduction rate were determined to be 504 and 819 mg/kg SDW, respectively.
All validity criteria recommended by the test guidelines were fulfilled.
Executive summary:

Effects of 1,2-Benzenedicarboxylic acid,di-C8-10-alkyl esters on mortality, biomass and the reproductive potential of the earthworm species Eisenia fetida (Annelida, Lumbricidae) were determined according to OECD 222 (2004).

The study was conducted under static conditions over 8 weeks with the test item concentrations 20 – 62.5 – 125 – 250 – 500 – 1000 mg/kg SDW. Each application rate was mixed into artificial soil containing 10 % peat. A control using untreated artificial soil was tested under the same conditions as the test item treatments. 80 test organisms were inserted into 8 control replicates and 40 test organisms were divided into 4 replicates for each treatment (10 earthworms per replicate). They had an individual body weight between 0.40 and 0.54 g at the experimental start.

Each concentration level and the control were analysed via LC-MS analysis after preparation at the start of the exposure (day 0) and at the end of the exposure (day 56).

The measured concentrations of all active ingredients of the test item were within the range of 78 to 146% of the nominal values at the start of the exposure and within the range of 16 to 36 % at the end. For the test item 3 components were analytical verified. One of them, the C10C10 part, results in varying recovery’s. For the other components the results were homogeneous. Therefore the nominal test item concentrations were used for EC calculations.

After 28 days of exposure in soil, neither earthworm mortalities nor significant pathological symptoms or changes in the behaviour of adult earthworms were observed in the control or test item treatments.

There were no statistically significant differences in earthworm body weights in all treatment rates compared to the control. After a further four weeks, the reproduction rate (average number of juveniles produced) was 118 in the control and ranged from 88 to 126 in the test item treatment rates. Compared to the control, no statistically significant reduction of reproduction rate was observed in the test item concentrations 20 to 500 mg/kg SDW. However, at the test item concentration 1000 mg/kg SDW the earthworm reproduction was statistically significantly reduced. Overall, the NOEC of the test item concerning mortality, biomass and reproduction was determined to be 500 mg/kg SDW. The EC10/20-values for reproduction rate were determined to be 504 and 819 mg/kg SDW, respectively.

All validity criteria recommended in the test guideline were fulfilled.

Description of key information

(Short-term) LC50 > 1000 mg/kg dry soil

(Long-term) NOEC = 500 mg/kg dw, EC10 = 504 mg/kg dw

Key value for chemical safety assessment

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

Additional information

In the Eisenia fetida study with 1,2 -Benzenedicarboxylic acid, di-C8 -10 -alkyl esters after 7 and 14 d no mortality up to the highest concentration of 1000 mg/kg was observed. This study is supported by the result with the structurally related substance 1,2-Benzenedicarboxylic acid, di-C6-10-alkyl esters with a LC10 in excess of 1000 mg/kg dry soil.

In the chronic earthworm study with 1,2 -Benzenedicarboxylic acid, di-C8 -10 -alkyl esters, no mortalities, significant pathological symptoms or changes in the behaviour of adult earthworms were observed in the control or any test item treatments. The reproduction was significantly affected at 1000 mg/kg SDW and therefore the lowest NOEC of the test item based on reproduction was determined to be 500 mg/kg SDW.