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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:
14/10/2015-11/02/2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Reliability 1 is assigned because the test has been carried out following the international guideline and under GLP without deviations that influence the results
Qualifier:
according to
Guideline:
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
Qualifier:
according to
Guideline:
ISO 11268-2 (Effects of Pollutants on Earthworms. 2. Determination of Effects on Reproduction)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Details on sampling:
From each test item group and of the control two specimens of about 20 g of treated substrate were sampled, and stored deep frozen.
Vehicle:
yes
Details on preparation and application of test substrate:
Artificial soil:
- 10% Sphagnum-peat, air-dried and finely ground (2 mm with no visible plant remains)
- 20% Kaolin clay (Kaolinite content >30%)
- 69.6% fine quarts-sand (F34) containing more than 50% by mass of particle size 0.05 mm to 0.2 mm.
- 0.4% Calcium carbonate (CaCO3) was added to adjust pH to 6.0 +/- 0.5

The artificial soil was moistened to approximately half of the final water content 1 day before the application. The additional water required to achieve the final water content was added when applying the test item.

Maximum WHC of the Articifial soil was 54% of the dry weight of artificial soil.
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
Adult worms of approximately 7 months, with well-developed clitellum. Age range between test individuals not differing by more than 4 weeks. Body weights ranged between 306 and 594 mg. Bred under standardised conditions in Ibacon laboratories in a breeding medium of cattle manure, peat, sand , calcium carbonate and straw, fed with catlle manure, stored at RT. Worms were acclimatised for 1 day in artificial soil under test conditions.
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
8 wk
Remarks:
Adults removed after 4 wks, offspring remained in test containers for another 4 weeks
Test temperature:
18 - 22 degrees Celsius.
pH:
At start of study pH 6.3 - 6.5
After 8 wks pH 5.6 - 6.4
Moisture:
At experimental start: 27.4% to 28.9% (50.7% to 53.6% of the maximum WHC, i.e. within the recommended range 40-60% of the total WHC)
At experimental end: 26.6% to 33.0% (49.3% to 61.1% of the maximum WHC)
Details on test conditions:
Controlled environment room, in a ventilated area.
Light intensity was within the range of 400 lux to 800 lux.
Light regime: 16 h light and 8 h dark
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 wass less than 10%.
Nominal and measured concentrations:
A stock solution was prepared by dissolving 1738 mg of Iso E Super in 25 mL acetone. A sequential dilution series was prepared to achieve following nominal concentrations: 3.16, 10, 31.6, 100 and 316 mg test item/kg soil. Then 10 mL of the corresponding dilutions were added to 20g fine quartz sand. The treated sand was left for approximately four hours in a fume hood until the solvent has evaporated and was mixed with a spoon. Then artificial soil equivalent to 2180 g dry weight was added to test item blended sand.
Samples were taken in case analyses would be considered to be necessary. No analyses were carried out.
Reference substance (positive control):
yes
Remarks:
Carbendazim
Duration:
28 d
Dose descriptor:
LC50
Effect conc.:
270.5 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: mortality
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
100 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: mortality
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
31.6 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: body weight
Key result
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
31.6 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
See also tables at 'any other informtion on results incl. tables'.
Mortality of adult worms:
A statistically significantly mortality of 77.5% was observed at the highest concentration of 316 mg test item/kg soil (Fisher's Exact Test, one-sided greater, alpha = 0.05). No additional mortality was observed.

Body weights of adult worms:
The bw changes in the test item treated groups were not statistically significantly different compared to the control group up to and including the test concentration of 31.6 mg test item/kg soil. At 100 mg test item/kg soil the weight increase was statistically significantly reduced compared to the control. The concentration of 316 mg test item/kg soil was not evaluated due to the low number of surviving worms.

Reproductive assessment:
The reproduction rates were not statistically significanly different compared to the control up to and including the test concentration of 31.6 mg test item/kg soil. At 100 mg test item/kg soil and above reproduction was statistically significantly reduced compared to the control.

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 up to the concentration of 100 mg test item/kg soil whereas the food intake at the concentration of 316 mg test item/kg soil appeared to be reduced.

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):
In a test with reference item Carbendazim 600 g/L SC (June to August 2015) there were statistically significant effects on reproduction at a concentration of 2.08 mg a.i./kg soil and higher, which is in line with the guideline OECD 222 (effects should be observed between 1 and 5 mg a.i./kg soil). The EC50 for reproduction was calculated as 1.91 mg a.i./kg soil.
Reported statistics and error estimates:
Mortality data were analysed for significance by using the Fisher's Exact Binomial Test (multiple comparison, with Bonferroni correction, alpha = 0.05, one-sided greater).
The body weight change and reproduction data were tested for normal distribution and homogeneity of variance (alpha = 0.05) using the Shapiro-Wilk's test and the Levene's test, respectively.
As the data for body weight changes were normally distributed but not homogeneous the Bonferroni-Welch t-test was used to compare treatment and control values (multiple comparison, two-sided alpha = 0.05).
As the data for reproduction were normally distributed and homogeneous the Williams t-test was used to compare treatment and control values (multiple comparison, one-sided alpha = 0.05).
The LC50 and the EC10 and EC20 values for reproduction were calculated by applying Probit-Analysis.

Table - Effect of Iso E Super on earthworms (Eisenia fetida) in a 56 -day reproduction study

 Iso E Super[mg/kg soil dw] Control  3.16  10.0  31.6  100  316 
Mortality (day 28) [%]  77.5* 
 Body weight change (day 28) [%] 23.0  22.5  22.0  14.7  9.0*  -8.1 
 Mean No. of juveniles (day 56) 183  185  181  171  127* 
 Reproduction in [%] of control (day 56) 101  98.4  93.0  69.4*
 Food consumption [g] 25.0  25.0  25.0  25.0  25.0  7.5* 

 * statistically significant.

 

Validity criteria fulfilled:
yes
Remarks:
Control mortality was 0% and so this validity criterion was met. The number of juvenile worms per replicate was 126 to 227 and so this validity criterion was met. The coefficient of variation of reproduction in control was 16.4% and thus met.
Conclusions:
4wk-NOEC (mortality) = 100 mg/kg DW; 4wk-NOECbody weight = 31.6 mg/kg DW; 8 wk-NOECreproduction = 31.6 mg/kg DW.
Executive summary:

The earthworm Eisenia foetida was tested. The test material at appropriate concentrations were dissolved in equal amounts of acetone, mixed with the quartz sand and allowed to slowly evaporate and mixed with the standard soil  containing 10% Sphagnum peat, 20% kaolinite clay, approximately 70% fine quartz-sand (grain size 0.05 -0.2 mm) and 0.4% calcium carbonate to adjust to pH 6.0±0.5. The earthworm test was carried out according to OECD 222 and ISO 11268. Adult worms (Eisenia fetida) were exposed to nominal concentrations in soil of 3.16, 10, 31.6, 100 and 316 mg/kg. Weights of the adult worms ranged between 306 and 594 mg. The worms were fed weekly with finely ground cattle manure. Adult worms were removed after 4 weeks of exposure, counted and weighed. The remaining offspring remained in the test containers for another four weeks. Mortality of the adults was not affected after 4 weeks in concentrations up to 100 mg/kg. Body weight was significantly reduced in the 100 mg/kg group. Reproduction was significantly affected at a concentration of 100 mg/kg (30.6% inhibition, LOEC). In the highest concentration of 316 mg/kg reproduction was not assessed because of the low survival rate of adults.

Description of key information

See below.

Key value for chemical safety assessment

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

Additional information

The earthworm Eisenia foetida was tested.The test material at appropriate concentrations were dissolved in equal amounts of acetone, mixed with the quartz sand and allowed to slowly evaporate and mixed with the standard soil containing 10% Sphagnum peat, 20% kaolinite clay, approximately 70% fine quartz-sand (grain size 0.05 -0.2 mm) and 0.4% calcium carbonate to adjust to pH 6.0±0.5.The earthworm test was carried out according to OECD 222 and ISO 11268. Adult worms (Eisenia fetida) were exposed to nominal concentrations in soil of 3.16, 10, 31.6, 100 and 316 mg/kg. Weights of the adult worms ranged between 306 and 594 mg. The worms were fed weekly with finely ground cattle manure. Adult worms were removed after 4 weeks of exposure, counted and weighed. The remaining offspring remained in the test containers for another four weeks. Mortality of the adults was not affected after 4 weeks in concentrations up to 100 mg/kg. Body weight was significantly reduced in the 100 mg/kg group. Reproduction was significantly affected at a concentration of 100 mg/kg (30.6% inhibition, LOEC). In the highest concentration of 316 mg/kg reproduction was not assessed because of the low survival rate of adults.