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Ecotoxicological information

Toxicity to soil macroorganisms except arthropods

Administrative data

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:
other: Study acccording to OECD protocol under GLP, no deviations with impact on test results, validity criteria met.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Substance type ethylene amine, N=5:
- Physical state: liquid
- Analytical purity: see section 1.3
- Impurities (identity and concentrations): see section 1.3
- Composition of test material, percentage of components: see section 1.3
- Isomers composition: see section 1.3
-Test item: Tetraethylenepentamine, TEPA
-Batch number: P0981
-Chemical name: Tetraethylenepentamine, linear, cyclic and branched
-CAS No.: 90640-66-7
-EC No.292-587-7
-Purity (certified): 96.2 % (TEPA fraction)
-Density: 0.991 – 0.999 g/cm3 (20 °C)
Appearance: offwhite, clear, liquid
-Solubility (in water at RT): > 1000 g/L
-Recommended storage: Protected from direct sunlight, in the tightly closed, original container in a dry, cool and well-ventilated area.


Sampling and analysis

Analytical monitoring:
no

Test substrate

Vehicle:
no
Details on preparation and application of test substrate:
- Method of application to filter paper (if used):
- Method of mixing into soil (if used):
- Method of application to soil surface (if used):
- Controls:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
- Concentration of vehicle in test medium (stock solution and final test solution):
- Evaporation of vehicle before use:

Test organisms

Test organisms (species):
Eisenia fetida
Details on test organisms:
Test System Eisenia fetida (Annelida, Lumbricidae)

Reason for the selection Eisenia fetida is suitable for this kind of study and is one of the recommended species according to the guideline.

Source Breeding stock culture maintained at the test facility

Breeding Organisms of the species Eisenia fetida are bred at the test facility in covered plastic vessels containing potting compost.

Synchronisation of the Earthworms of homogeneous age and body weight were used in
earthworm population the definitive test. Synchronisation of the population was achieved by placing adult earthworms into breeding boxes and removing the adults after 4 weeks. Offspring from the remaining cocoons reached the adult age after 2 months at the earliest.

Feeding During breeding, the earthworms were fed with a litter of dried stinging nettle leaves and porridge oats. A sufficient amount of the food was provided depending on the feeding rate and the density of the earthworm population in the vessels.
Test duration 8 weeks

Number of replicates 8 replicates per control and 4 replicates per test item concentration.

Number and age of 10 adult earthworms (with clitellum) aged 2-12 months were used
the earthworms per replicate. The difference of age did not deviate by more than 1 month (see “Synchronisation”).

Body weight (actual) 0.31 to 0.52 g at the start of exposure

Adaptation Earthworms were adapted to untreated artificial soil two days prior to the test start.

Homogeneity of groups The worms were washed with demineralised water and dried gently on a paper towel. The homogeneity of the population was checked by weighing the earthworms. After confirmation of homogeneity, groups of 10 earthworms (chosen by a randomized procedure) were weighed and introduced to each test vessel.



Study design

Study type:
laboratory study
Substrate type:
natural soil
Limit test:
yes
Total exposure duration:
556 d
Post exposure observation period:
not applikcable

Test conditions

Test temperature:
Table 21: Environmental Conditions
Room Temperature [°C] Photoperiod [h] Light intensity [lx]
nominal 20  2 16 400 - 800
actual 18 – 23* 586 ± 107
* The temperature was above 22 °C for 6 hours in total.

pH:
Table 20: pH-Values, Moisture and WHCmax of the Test Media
Test item concentration
[mg/kg soil dry weight] pH-value Moisture [%] of dry weight WHCmax of artificial soil
[g/100 g DW]
day 0 day 56 day 0 day 56
Control 6.19 6.21 32.5 35.2 57.1
62.5 5.95 6.06 31.9 35.1
125 5.97 6.26 30.4 32.7
250 6.05 6.28 29.7 33.8*
500 6.15 6.30 29.7 31.3
1000 6.28 6.37 29.1 33.3*
*) Soil moisture slightly deviated by more than 10% from the initial value. This deviation is considered to have no
impact on quality and integrity of the study.
WHC = Water Holding Capacity
Moisture:
Table 20: pH-Values, Moisture and WHCmax of the Test Media
Test item concentration
[mg/kg soil dry weight] pH-value Moisture [%] of dry weight WHCmax of artificial soil
[g/100 g DW]
day 0 day 56 day 0 day 56
Control 6.19 6.21 32.5 35.2 57.1
62.5 5.95 6.06 31.9 35.1
125 5.97 6.26 30.4 32.7
250 6.05 6.28 29.7 33.8*
500 6.15 6.30 29.7 31.3
1000 6.28 6.37 29.1 33.3*
*) Soil moisture slightly deviated by more than 10% from the initial value. This deviation is considered to have no
impact on quality and integrity of the study.
WHC = Water Holding Capacity

Details on test conditions:
Number of replicates 8 replicates per control and 4 replicates per test item concentration.
Number and age of 10 adult earthworms (with clitellum) aged 2-12 months were used
the earthworms per replicate. The difference of age did not deviate by more than 1 month (see “Synchronisation”).
Test medium 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)
- 0.40 % calcium carbonate (CaCO3) to achieve a pH of 6.0  0.5
The maximum water holding capacity, the moisture content and the pH of the artificial soil were determined. Two days before the test start the artificial soil (20 kg) was pre-moistened by adding 2280 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.
Test containers Test units containing about 600 g soil dry weight were 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. Transparent and perforated lids enabled sufficient gas exchange, light input and prevented the test substrate from drying.
Application The appropriate test item quantity was weighed out for each test item concentration and dissolved in demineralised water.
The spiking solutions and additional demineralised water were added to the artificial soil to adjust the humidity of the artificial soil to a moisture of 54 % of the maximum water holding capacity. Subsequently, the test medium was thoroughly mixed to ensure a homogenous distribution and about 600 g soil dry weight equivalent were filled into test vessels.
Nominal and measured concentrations:
Test concentrations: 0.5 – 1 – 2 – 4 mg a.i./kg soil dry weight (nominal)

Reference substance (positive control):
yes
Remarks:
Reference Item CARBENDAZIM Time schedule The test is conducted once every twelve months. The most recent test was carried out from February 21th to April 22th, 2014 (Study No. RBN1402).

Results and discussion

Effect concentrationsopen allclose all
Duration:
55 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:
55 d
Dose descriptor:
NOEC
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Duration:
55 d
Dose descriptor:
NOEC
Effect conc.:
ca. 125 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
55 d
Dose descriptor:
LOEC
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
55 d
Dose descriptor:
LOEC
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Duration:
55 d
Dose descriptor:
LOEC
Effect conc.:
ca. 250 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
55 d
Dose descriptor:
EC10
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: confidence limits were corrected for heterogeneity and are therefore not reported
Duration:
55 d
Dose descriptor:
EC10
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks on result:
other: confidence limits were corrected for heterogeneity and are therefore not reported
Duration:
55 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: confidence limits were corrected for heterogeneity and are therefore not reported
Duration:
55 d
Dose descriptor:
EC10
Effect conc.:
ca. 167 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: confidence limits were corrected for heterogeneity and are therefore not reported
Duration:
55 d
Dose descriptor:
other: EC20
Effect conc.:
ca. 360 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: confidence limits were corrected for heterogeneity and are therefore not reported
Details on results:
- Mortality at end of exposure period: none
- Total mass of adults at beginning of test:Body Weight Changes of the Adult Earthworms and - Changes in body weigth of live adults (% of initial weight) at end of exposure period:
Test item concentration
[mg/kg soil dry weight] Replicate Mean body weights per replicate Mean body weight gain of earthworms
Test start 28 days Per replicate Mean ± SD Sig.
[g] [g] [g] [%] [g] [%]
Control 1 0.40 0.50 0.10 25.0 0.13 ± 0.03 33.0 ± 7.81 –
2 0.40 0.51 0.11 27.5
3 0.40 0.53 0.13 32.5
4 0.40 0.57 0.17 42.5
5 0.41 0.51 0.10 24.4
6 0.41 0.59 0.18 43.9
7 0.41 0.53 0.12 29.3
8 0.41 0.57 0.16 39.0
62.5 1 0.41 0.54 0.13 31.7 0.12 ± 0.01 28.9 ± 4.03 No
2 0.42 0.52 0.10 23.8
3 0.40 0.51 0.11 27.5
4 0.40 0.53 0.13 32.5
125 1 0.41 0.53 0.12 29.3 0.13 ± 0.01 30.9 ± 3.24 No
2 0.40 0.54 0.14 35.0
3 0.40 0.51 0.11 27.5
4 0.41 0.54 0.13 31.7
250 1 0.41 0.57 0.16 39.0 0.14 ± 0.03 35.2 ± 8.84 No
2 0.40 0.53 0.13 32.5
3 0.40 0.58 0.18 45.0
4 0.41 0.51 0.10 24.4
500 1 0.40 0.56 0.16 40.0 0.16 ± 0.01 38.3 ± 1.52 No
2 0.41 0.56 0.15 36.6
3 0.41 0.57 0.16 39.0
4 0.40 0.55 0.15 37.5
1000 1 0.40 0.57 0.17 42.5 0.17 ± 0.05 41.5 ± 11.7 No
2 0.41 0.55 0.14 34.1
3 0.40 0.63 0.23 57.5
4 0.41 0.54 0.13 31.7
SD = Standard deviation Sig. = Statistical Significance (Williams Multiple Sequential t-test) compared to control


- No. of offspring produced: Reproduction Rate (Number of Juveniles after eight weeks)
Test item concentration
[mg/kg soil dry weight] Replicate Number of juveniles Mean ± SD CV [%] [%]
Reduction relative to the control Sig.
Control 1 141 144 ± 27.0 18.8 - -
2 149
3 142
4 162
5 119
6 95
7 177
8 169
62.5 1 137 152 ± 19.1 12.6 0 No
2 175
3 159
4 135
125 1 135 156 ± 16.5 10.6 0 No
2 150
3 168
4 170
250 1 113 120 ± 11.9 9.88 17.2 Yes
2 135
3 122
4 108
500 1 93 94 ± 6.90 7.34 34.7 Yes
2 100
3 85
4 99
1000 1 89 95 ± 21.2 22.3 34.3 Yes
2 105
3 68
4 117
SD = Standard deviation CV = Coefficient of variation
Sig. = Statistical Significance (Williams Multiple Sequential t-test) compared to the control

- No. of unhatched cocoons: not provided
- Morphological abnormalities: none
- Behavioural abnormalities: none
- Other biological observations: nothing remarkabke
Results with reference substance (positive control):
14 Data of Reference Item

Table 22: Reference Item - Reproduction Rate (Number of Juveniles after 56 days)
Application rate
[mg a.i./kg DW] Rep. Number of juveniles Mean ± SD CV % of control Significance*
Control 1 78 72 ± 18.9 26.2 _ _
2 71
3 102
4 47
5 71
6 45
7 75
8 86
0.5 1 79 95 ± 11.3 11.9 132 No
2 94
3 105
4 100
1 1 106 95 ± 12.0 12.6 132 No#
2 79
3 93
4 102
2 1 34 52 ± 12.7 24.4 72.2 No
2 54
3 58
4 63
4 1 0 1 ± 0.58 58.0 1.39 Yes
2 0
3 1
4 1
Rep. = Replicate a.i. = active ingredient SD = Standard deviation CV = Coefficient of variation
*) = Statistically significantly reduced compared to control (t-Test)
#) = Statistically significant but not dose-dependent promotion of reduction

According to OECD 222 significant effects of Carbendazim should be observed between 1 and 5 mg a.i./kg soil DW.
Reported statistics and error estimates:
see attachted doc

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
This study on the effects of Tetraethylenepentamine, TEPA on the reproduction of earthworms of the species Eisenia fetida was performed according to OECD Guideline 222 (2004)
Conclusions:
Under the conditions of this study, Tetraethylenepentamine, TEPA did not induce significant mortality, pathological symptoms or changes of the behaviour of adult earthworms after 28 days of exposure at all tested concentrations. The adult earthworm biomass increased in the control and at all test item concentrations without any statistically significant differences. After a further four weeks of exposure the numbers of juvenile earthworms were statistically significantly lower at the test item concentrations 250, 500 and 1000 mg test item/kg soil dry weight than in the untreated control.

Thus, the No Observed Effect Concentration (NOEC) of Tetraethylenepentamine, TEPA summarizing the endpoints mortality, growth and reproduction of Eisenia fetida after eight weeks of exposure in artificial soil was determined to be 125 mg test item/kg soil dry weight. The EC10-value for reproduction was determined to be 167 mg test item/kg soil dry weight and the EC20-value was 360 mg test item/kg soil dry weight.
Executive summary:

 Summary

Effects ofTetraethylenepentamine, TEPA(Batch no.P0981) on mortality, growth and the reproductive potential of the earthworm speciesEisenia fetida(Annelida, Lumbricidae) were determined according to OECD Guideline 222 (2004) fromSeptember 11th to November 6th, 2014with the definitive exposure phase from September 11th to November 5th, 2014 atDr.U.Noack-Laboratorienin D-31157 Sarstedt, Germany. The study was conducted over 8 weeks with test item concentrations of 62.5, 125, 250, 500 and 1000 mg/kg soil dry weight, which were mixed into artificial soil containing 10 % peat. A control using untreated artificial soil was also set up.Sexually mature earthworms were inserted into 8 control replicates and into 4 replicates per test item concentration (10 earthworms per replicate).They had an individual body weight between 0.31 and 0.52 g at the start of exposure

After 28 days of exposure in soil, no evident earthworm mortalities, pathological symptoms or changes in the behaviour of adult earthworms were observed in all treatments. Moreover, no statistically significant differences in growth (earthworm body weight gain) were observed at all test item concentrations compared to the control. After a further four weeks the reproduction rate (average number of juveniles produced per replicate) was 144 in the control and ranged from 94 to 156 in the test item treatment groups, with the size of the reduction increasing in response to dose. Compared to the control, a statistically significant reduction of reproduction was observed in the test item concentrations 250, 500 and 1000 mg/kg soil dry weight. Consequently, the NOEC of the test item concerning reproduction was determined to be 125 mg/kg soil dry weight.The EC50-values for reproduction and growth could not be calculated reliably since no reduction ≥ 50 % for reproduction and growth occurred, however they exceeded 1000 mg test item/kg soil dry weight, the highest concentration tested. The EC10-value for reproduction was determined to be 167 mg test item/kg soil dry weight and the EC20-value was 360 mg test item/kg soil dry weight. The confidence limits were corrected for heterogeneity and are therefore not reported. All validity criteria specified by the test guidelines were fulfilled.