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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:
31 Jul 2014 - 03 Feb 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on preparation and application of test substrate:
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
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: earthworm (Eisenia fetida)
- Source: breeding stock culture maintained at the test facitity
- Age at test initiation (mean and range, SD): 2-12 months
- Weight at test initiation (mean and range, SD): 0.34-0.56 g at start of exposure

ACCLIMATION
- Acclimation period: 2 days
- Acclimation conditions (same as test or not): untreated artificial soil as used in the test
- Health during acclimation (any mortality observed): no mortality
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
56 d
Post exposure observation period:
exposure of adults: 28 days. exposure of offspring an additional 28 days . No post-exposure observations
Test temperature:
20 +- 2 °C
pH:
The initial pH value of the artificial soil was 6.16 (measured on day -6). Moisture content of the test medium as well as pH-values at test start and end are given in Table 21. Temperature during the study and light intensity are given in Table 22.

Table 21: pH-Values, Moisture and WHCmax of the Test Media
Application rate
[mg test item/
kg soil dry weight] pH-value Moisture [%] of dry weight WHCmax of artificial soil
[g/100 g DW]
day 0 day 57 day 0 day 56
Control 6.05 6.56 27.9 30.8* 52.2
62.5 5.94 6.34 26.9 31.8*
125 5.97 6.46 26.6 32.9*
250 6.13 6.40 27.0 32.5*
500 6.22 6.53 27.0 33.5*
1000 6.39 6.37 27.2 33.6*
*) Soil moisture deviated by slightly 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:
The initial pH value of the artificial soil was 6.16 (measured on day -6). Moisture content of the test medium as well as pH-values at test start and end are given in Table 21. Temperature during the study and light intensity are given in Table 22.

Table 21: pH-Values, Moisture and WHCmax of the Test Media
Application rate
[mg test item/
kg soil dry weight] pH-value Moisture [%] of dry weight WHCmax of artificial soil
[g/100 g DW]
day 0 day 57 day 0 day 56
Control 6.05 6.56 27.9 30.8* 52.2
62.5 5.94 6.34 26.9 31.8*
125 5.97 6.46 26.6 32.9*
250 6.13 6.40 27.0 32.5*
500 6.22 6.53 27.0 33.5*
1000 6.39 6.37 27.2 33.6*
*) Soil moisture deviated by slightly 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:
TEST SYSTEM
- Test container (material, size): round plastic boxis , inner diameter 15 cm, height 14 cm, transparent and perforated lids
- Amount of soil or substrate: 600 gr. dry 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 vehicle control: not applicable

TEST MEDIUM
Artificial soil, composed of 10% peat, 20 Kaolin, 69% air dried sand with particle sice of 0,05-0,2 mm; o,4% calcium carbonate to reach a pH of 6.0 +- 0.5


OTHER TEST CONDITIONS
- Photoperiod: 16 hr/d
- Light intensity: 400-880 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
3.2.4 Type and Frequency of Measurements and Observations

Measurements and observations : At the start and end of exposure, pH-value and moisture content of the test medium were determined in every treatment and control from pooled samples of all replicates. The water content of each test container was checked weekly by weighing. The weight loss was replenished with the appropriate amount of demineralised water. On days 7, 14, 21 and 28, additional 10 mL of demineralised water were added as the soil appeared to be too dry. The body weight of the adult earthworms was individually determined on day 0 and day 28 for each replicate.
After the first four weeks the adult earthworms were removed. Mortality and morphological changes of the adult earthworms were recorded for each test container.
After a further four weeks, the number of offspring hatched from the cocoons was counted for each test container.


Observation parameters
A) no obvious pathological symptoms
B) no reaction to touching
C) no negative phototactical reaction
D) spontaneous segmentation and separation
E) spasmodic winding
F) yellow secretion from the oral aperture
G) ulceration or bleeding of the skin
H) dead earthworms

Equipment pH-Meter, Multi 340i, WTW
Hygrothermograph, LUFFT
Drying cabinet, BINDER GmbH
Analytical balances, SARTORIUS
Balances, KERN
Balances, WEDO
Waterbath, MEMMERT
Automatic Timer
Mixer, SEVERIN
Luxmeter, TESTO GMBH


VEHICLE CONTROL PERFORMED: not applicable

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2 (from 62.5 upwards)
- Range finding study
- Test concentrations: Control -1 -10 -100- 1000 mg/kg
- Results used to determine the conditions for the definitive study: no mortality, no significant changes in body weight, no clinical signs
Nominal and measured concentrations:
Nominal concentrations: Application rates 62.5 – 125 – 250 – 500 – 1000 mg test item/kg soil dry weight
Measured concentrations: not measured
Reference substance (positive control):
yes
Remarks:
Carbendazim, annually performed . Most recent test Febr 11-April 22n 2014 . results as expected
Duration:
56 d
Dose descriptor:
EC10
Effect conc.:
ca. 31.1 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 95% CI: <62.5 - 67 mg/kg dw
Duration:
56 d
Dose descriptor:
other: EC20
Effect conc.:
ca. 86.62 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 95% CI: < 62.5-188 mg/kg dw
Duration:
56 d
Dose descriptor:
EC50
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.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks on result:
other: no effect on growth at any concentration up to and including 1000 mg/kg soil
Duration:
56 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: no mortality at any concentration up to and including 1000 mg/kg soil
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
ca. 62.5 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
ca. 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
ca. 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Details on results:
No significant mortality of adult earthworms was observed in the control or at any of the test item concentrations after 28 days of exposure to the test item (Table 5). Therefore, no ECx-values could be determined.

Table 5: Mortality [%] of Adult Earthworms after 28 Days of Exposure
Test item concentration Replicate
[mg test item/kg soil dry weight] 1 2 3 4 5 6 7 8 Mean
Control 0 0 0 0 0 0 0 0 0
62.5 0 0 0 0 – 0
125 0 0 0 0 0
250 0 10 0 0 2.5
500 0 0 0 0 0
1000 0 0 0 0 0




7.2.2 Pathological Symptoms and Changes in Behaviour
No pathological symptoms nor changes in the behaviour of the earthworms were observed in the control or in any of the test item concentrations (Table 6).

Table 6: Earthworm Behaviour and Pathological Symptoms after 28 Days of Exposure
Test item concentration Replicate
[mg test item/kg soil dry weight] Observation parameter 1 2 3 4 5 6 7 8
Control A 10/10 10/10 10/10 10/10 10/10 10/10 10/10 10/10
62.5 A 10/10 10/10 10/10 10/10 –
125 A 10/10 10/10 10/10 10/10
250 A
H 10/10
- 9/10
1/10 10/10
- 10/10
-
500 A 10/10 10/10 10/10 10/10
1000 A 10/10 10/10 10/10 10/10
A) No visible symptoms or behavioural changes H) dead


7.2.3 Live Weight
At the test start the earthworms had individual weights of 0.34 - 0.56 g, which are consistent with the range recommended by the test guideline. The biomass of the adult earthworms increased throughout the first 28 days at all application rates, with no statistically significant differences (ANOVA, α = 0.05) compared to the control (Table 7). Therefore, no ECx-values could be determined. The individual body weights are given in Table 9 to 20.














Table 7: Body Weight Changes of the Adult Earthworms
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 0.43 0.54 0.11 25.6 0.14 ± 0.02 31.2 ± 4.99 –
2 0.44 0.61 0.17 38.6
3 0.45 0.62 0.17 37.8
4 0.44 0.57 0.13 29.5
5 0.44 0.57 0.13 29.5
6 0.44 0.58 0.14 31.8
7 0.44 0.58 0.14 31.8
8 0.44 0.55 0.11 25.0
62.5 1 0.45 0.57 0.12 26.7 0.13 ± 0.02 29.8 ± 3.47 No*
2 0.44 0.56 0.12 27.3
3 0.44 0.59 0.15 34.1
4 0.45 0.59 0.14 31.1
125 1 0.44 0.57 0.13 29.5 0.16 ± 0.03 36.3 ± 5.66 No*
2 0.44 0.59 0.15 34.1
3 0.43 0.60 0.17 39.5
4 0.45 0.64 0.19 42.2
250 1 0.44 0.56 0.12 27.3 0.16 ± 0.03 36.2 ± 6.14 No*
2 0.44 0.62 0.18 40.9
3 0.43 0.59 0.16 37.2
4 0.43 0.60 0.17 39.5
500 1 0.44 0.58 0.14 31.8 0.16 ± 0.02 36.8 ± 5.39 No*
2 0.44 0.59 0.15 34.1
3 0.43 0.62 0.19 44.2
4 0.43 0.59 0.16 37.2
1000 1 0.44 0.52 0.08 18.2 0.14 ± 0.04 32.0 ± 9.41 No*
2 0.43 0.59 0.16 37.2
3 0.44 0.59 0.15 34.1
4 0.44 0.61 0.17 38.6
*) compared to control
SD = Standard deviation Sig. = Statistical Significance (ANOVA, α = 0.05)

7.2.4 Reproduction Rate

The mean number of juveniles per replicate in the control was 190. The average number of juveniles per replicate in the treated groups ranged from 116 to 158 (Table 8, Figure 1). There was a dose-related reduction (16.8 to 39.0 %, compared to the control) in the number of juveniles produced across the range of test item treatments, and the reproduction of the earthworms was statistically significantly different from the control at the test item concentrations 125 – 1000 mg test item/kg soil dry weight. The EC50-value was not determined since no reduction of reproduction > 50 % occurred, but it exceeded 1000 mg test item/kg soil dry weight, the highest concentration tested. The EC10 was extrapolated to 31.1 mg test item/kg soil dry weight (below the lowest concentration tested) and the EC20-value was determined to be 86.6 mg test item/kg soil dry weight. The coefficient of variation calculated for the reproduction of the control was 15.0 % and was thus lower than 30 %.



Figure 1: Reproduction after Eight Weeks of Exposure

*) = statistically significantly different from control



















Table 8: Reproduction Rate (Number of Juveniles after 8 Weeks)
Test item concentration
[mg /kg soil dry weight] Replicate Number of juveniles Mean ± SD CV [%] [%]
Reduction relative to the
control Sign.*
Control 1 166 190 ± 28.5 15.0 - -
2 155
3 207
4 232
5 177
6 176
7 227
8 181
62.5 1 152 158 ± 19.5 12.4 16.8 No
2 165
3 181
4 135
125 1 155 145 ± 8.14 5.61 23.7 Yes
2 136
3 142
4 148
250 1 131 133 ± 5.56 4.18 30.0 Yes
2 135
3 139
4 126
500 1 127 130 ± 15.3 11.8 31.6 Yes
2 110
3 136
4 146
1000 1 152 116 ± 24.2 20.9 39.0 Yes
2 104
3 102
4 105
SD = Standard deviation CV = Coefficient of variation Sig. = Statistical Significance (ANOVA, α = 0.05)
*) compared to control


- Mortality at end of exposure period: no significant mortatlity
- Total mass of adults at beginning of test: ~123 gr
- Changes in body weigth of live adults (% of initial weight) at end of exposure period: contro;s 31.2%+- 4.99. 62.5 mg/kg soil dw: 29.8% +- 3.47; 125 mg/kg soil dw: 36,3% +- 5.66; 250 mg/kg soil dw: 36,2% +- 6.14; 500 mg/kg soil; dw: 36.8% +- 5.39; 1000 mg/kg soil dw: 32.0% +- 9.41. None of the differences are statistically significant
- No. of offspring produced: Controls: 190. exposed: dose related reducten (16.8 -to 39% )
- No. of unhatched cocoons: not given
- Morphological abnormalities :no morphological abnormailities observed
- Behavioural abnormalities:No behavioural abnormailites observed
Results with reference substance (positive control):
Data of Reference Item

Table 23: Reference Item - Reproduction Rate (Number of Juveniles after 8 weeks)
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 no dose-dependent promotion of reduction

According to the 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 attached document "statistical analyses.docx"
Validity criteria fulfilled:
yes
Remarks:
The adult mortality in the control group did not exceed 10% after the first four weeks of the definitive test. The average number of juveniles in the control group was higher than 30 per replicate. coef. of var,.for the reprod of controls < 30% at end
Conclusions:
Under the conditions of this study,Triethylenetetramine, TETA did not induce significant mortality, pathological symptoms or changes of the behaviour of adult earthworms after 28 days of exposure in all tested concentrations.The adult earthworm biomass increased in the control and at all application rateswithout 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 125 to 1000 mg test item/kg soil dry weight than in the untreated control. Thus, the No Observed Effect Concentration (NOEC) forTriethylenetetramine, TETA, summarizing the endpoints mortality, growth and reproduction of Eisenia fetida after 8 weeks of exposure in artificial soil was determined to be 62.5 mg test item/kg soil dry weight
Executive summary:

Effects of Triethylenetetramine, TETA (Batch no. P0968) on mortality, growth and the reproductive potential of the earthworm species Eisenia fetida (Annelida, Lumbricidae) were determined according to OECD Guideline 222 (2004) from August, 13th to October 9th, 2014 with the definitive exposure phase from August 13th to October 7th/8th, 2014 at Dr.U.Noack-Laboratorienin D-31157 Sarstedt, Germany. The study was conducted over 8 weeks with the test item concentrations 62.5, 125, 250, 500 and 1000 mg test item/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). The earthworms had an individual body weight between 0.34 and 0.56 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 in all test item concentrations compared to the control. After a further four weeks the reproduction rate (average number of juveniles produced per replicate) was 190 in the control and ranged from 116 to 158 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 at all test item concentrations from 125 to 1000 mg test item/kg soil dry weight. Consequently, the NOEC of the test item concerning reproduction of earthworms was determined to be 62.5 mg test item/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 31.10 mg test item/kg soil dry weight (a value extrapolated below the lowest test concentration) and the EC20-value was determined to be 86.62 mg test item/kg soil dry weight. All validity criteria specified by the test guidelines were fulfilled.

Description of key information

 NOEC (56 d): 62.5 mg/kg soil dw based on reproduction

Key value for chemical safety assessment

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

Additional information

The chronic toxicity of the substance to earthworms was tested according to OECD guideline 222 (GLP).

The test was conducted with artificial soil as substrate and adult specimen of Eisenia fetida as test organisms. The tested concentrations were 62.5, 125, 250, 500 and 1000 mg test item/kg soil dry weight. The total test period was 8 weeks. After 28 days of exposure the mortality as well as symptoms of intoxication of the adult animals was recorded. After an additional exposure period of 28 days the reproduction rate (number of juveniles) was determined. Under the conditions of this study the substance 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 125 - 1000 mg test item/kg soil dry weight than in the untreated control. Thus, a No Observed Effect Concentration (NOEC) of 62.5 mg test item/kg soil dry weight based on the reproduction rate was determined.