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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:
2019-02-28 to 2019-08-16 with the definitive exposure phase from 2019-06-21 to 2019-08-16
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))
Version / remarks:
2016
Deviations:
yes
Remarks:
Food was provided on day 0 (test start) instead of day 1 due to good experience with this procedure. At test end, the soil moisture deviated by more than 10 % from the initial value in all treatments.
GLP compliance:
yes
Specific details on test material used for the study:
Test item Sodium methyl oleoyl taurate
Batch number DEB4008582
Content (certified) 63.4%
CAS RN Not applicable
Appearance White powder
Water solubility 0.075 g/L (cmc)
Stability under Not specified
test conditions
Expiry date 2020-05-02
Recommended storage Room temperature 20°C
Analytical monitoring:
yes
Details on sampling:
On day 0 and at test end, the amount of test item in
soil was analytically verified via LC-MS/MS for the control and all test item concentrations using pooled samples of all respective replicates.
Vehicle:
no
Details on preparation and application of test substrate:
The respective test item amount for each test item concentration was weighed out, dispersed in demineralised water (200 mL) and added to the artificial soil (2440 g SDW). Additional demineralised water (156 mL) was added to adjust the humidity of the artificial soil to a moisture of 54 % of the WHCmax.
Subsequently, the test medium was thoroughly mixed to ensure a homogenous distribution and about 610 g SDW were filled into each test vessel.
Test organisms (species):
Eisenia fetida
Animal group:
annelids
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 earthworm population:
Earthworms of homogeneous age and body weight were used in 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 are 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.

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) 0.40 to 0.54 g

Adaption Earthworms were adapted to the artificial soil to be used for the test two days prior to 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 randomised procedure) were weighed and introduced into each test vessel.

Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
56 d
Test temperature:
18 - 22 °C
pH:
Application rate
[mg test item/kg SDW] pH-value
day 0 day 56
Control 6.72 6.17
62.5 6.73 6.14
125 6.69 6.17
250 6.79 6.10
500 6.71 6.17
1000 6.67 6.23


Moisture:
Application rate
[mg test item/kg SDW] Moisture [%] of dry weight

day 0 day 55
Control 27.5 31.3*
62.5 27.7 31.5*
125 27.8 30.9*
250 27.9 31.9*
500 27.6 31.3*
1000 27.5 31.1*

* = The 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.
Details on test conditions:
Test duration 8 weeks

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

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)
- 0.50 % calcium carbonate (CaCO3) to achieve a pH of 6.0 +- 0.5

The WHCmax, the moisture content and the pH-value of the artificial soil were determined and adjusted. Two days before test start the artificial soil (20000 g) was pre-moistened with 2305 g demineralised water to achieve a moisture content corresponding to 27 % of the WHCmax of the artificial soil. The moistened soil was kept covered to prevent evaporative water losses prior to the start of exposure.

Test container
Test units containing about 610 g SDW were round plastic boxes with an inner diameter of 15.0 cm corresponding to a bottom surface area of 177 cm2 and aheight of 14 cm. Transparent and perforated lids enable sufficient gas exchange, light input and prevent the test substrate from drying.

Photoperiod 16 h light and 8 h dark

Light intensity 400 - 800 lx

Feeding
The earthworms were fed with air-dried cattle manure weekly during the first 4 weeks as presented in Table 3. At test start and at day 28 the food was mixed carefully into the soil, on the other days the food was placed onto the soil surface. No further feeding took place during the remaining 4 weeks of the test
The cattle manure was delivered by LEHR- UND FORSCHUNGSGUT RUTHE, Schäferberg 1, 31157 Sarstedt, Germany.
Manure was obtained from cattle that were not subject to medication or treatment with substances, such as growth promoters, nematicides or similar veterinary products that could adversely affect the worms during the test. The manure was air-dried and finely ground before use.

Moistening
Throughout the test the replicates were weighed weekly to determine water loss via evaporation. Evaporated water was replenished by adding demineralised water to maintain the soil moisture which should not deviate by more than 10% of the initial value at the end of the test. Additional moistening was performed in case the soil appeared too dry.


Nominal and measured concentrations:
Based on the results of a preliminary study (see Annex II) five test item concentrations were tested::
Nominal: 1000 – 500 – 250 – 125 – 62.5 mg test item/kg SDW (factor 2)

Measured Concentrations and Percent of Nominal concentration of Sodium methyl oleoyl taurate
Sampling date Day 0 Day 55
Nominal concentration of the test item
[mg/kg SDW] Sodium methyl oleoyl taurate
Meas. conc.
[mg/kg SDW]
[%]
Meas. conc.
[mg/kg SDW]
[%]
1000 931 93 14.3 1
500 469 94 8.15 2
250 246 99 5.16 2
125 117 93 3.10 2
62.5 62.2 100 1.69 3
Control < LOQ < LOQ
Reference substance (positive control):
yes
Remarks:
CARBENDAZIM
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:
other: biomass
Key result
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:
other: biomass
Key result
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
250 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
56 d
Dose descriptor:
LOEC
Effect conc.:
500 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
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.:
>= 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:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
56 d
Dose descriptor:
other: EC20
Effect conc.:
587 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
56 d
Dose descriptor:
EC10
Effect conc.:
315 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
- No evident earthworm mortality (< 10%) was observed in the control and all test item concentrations
-At test start, the earthworms had individual weights of 0.40 - 0.54 g. There were no statistically significant differences in biomass in all treatment rates compared to the control.
-The reproduction rate (average number of juveniles produced) was 115 in the control and ranged from 86 to 133 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction at the treatment rates 62.5 to 250 mg test item/kg SDW compared to the control. However, at the test item concentrations 500 and 1000 mg test item/kg SDW the earthworm reproduction was statistically significantly reduced.
The coefficient of variation calculated for the reproduction of the control was 19.9 % and thus lower than 30 %.


Results with reference substance (positive control):
Reference Item - Reproduction Rate (Number of Juveniles after 8 Weeks)
Application rate
[mg a.i./kg
SDW] Rep. Number of juveniles Mean ± SD CV % of control Significance*
Control 1 137 105 ± 18.8 17.9 - -
2 96
3 131
4 105
5 98
6 87
7 96
8 90
0.5 1 109 90 ± 16.0 17.7 85.7 No
2 86
3 71
4 95
1 1 57 70 ± 12.2 17.4 66.7 Yes
2 79
3 63
4 82
2 1 87 78 ± 6.99 8.96 74.3 Yes
2 71
3 79
4 74
4 1 21 14 ± 8.19 58.5 13.3 Yes
2 20
3 8
4 5
Rep. = Replicate a.i. = active ingredient SD = Standard deviation CV = Coefficient of variation
*) = Statistically significantly reduced compared to control (Williams Multiple Sequential t-test Procedure)

According to OECD 222 significant effects of Carbendazim should be observed between 1 and
5 mg a.i./kg SDW.
Reported statistics and error estimates:
Adult Mortality The arithmetic mean and NOEC were determined. Since no evident mortality occurred the LCx – values were not determined.


Reproduction - Shapiro-Wilk’s Test on Normal Distribution
- Levene’s Test on Variance Homogeneity (with Residuals)
- Trend Analysis on Contrasts (Monotonicity of
Concentration/Response)
- William’s Multiple Sequential t-test Procedure

The a-value (acceptable probability of incorrectly concluding that there is a difference) was a = 0.05. For the endpoint of reproduction, the arithmetic mean and the variance (coefficient of variation) per treatment and control were calculated.
The EC10,20,50 - values for reproduction were calculated using 3-param. normal CDF.

Biomass - Shapiro-Wilk’s Test on Normal Distribution
- Levene’s Test on Variance Homogeneity (with Residuals)
- Trend Analysis on Contrasts (Monotonicity of
Concentration/Response)
- Dunnett’s Multiple Sequential t-test Procedure
The a-value (acceptable probability of incorrectly concluding that there is a difference) was a = 0.05. For the endpoint of biomass, the arithmetic mean and the variance (coefficient of variation) per treatment and control were calculated.

Mortality of Adult Earthworms in [%] after 28 Days of Exposure

Application rate       Replicate

[mg test item/kg  SDW]       1       2       3       4       5       6       7       8       Mean

Control       0       0       0       0       0       0       0       10       1.25

62.5       0       10       0       0       -       2.5

125       0       0       0       0              0

250       0       0       0       0              0

500       0       0       30       0              7.5

1000       0       0       0       10              2.5

Earthworm Behaviour and Pathological Symptoms after 28 Days of Exposure

Application rate       Replicate

[mg test item/kg  SDW]       Observation parameter       1       2       3       4       5       6       7       8

Control       A

H       10/10

-       10/10

-       10/10

-       10/10

-       10/10

-       10/10

-       10/10

-       9/10

1/10

62.5       A

H       10/10

-       9/10

1/10       10/10

-       10/10

-       -

125       A       10/10       10/10       10/10       10/10       

250       A       10/10       10/10       10/10       10/10       

500       A

H       10/10

-       10/10

-       7/10

3/10       10/10

-       

1000       A

H       10/10

-       10/10

-       10/10

-       9/10

1/10       

A = no obvious pathological symptoms       H = earthworm dead

Body Weight Changes of the Adult Earthworms       

Application rate

[mg test item/kg  SDW]       Replicate       Mean body weights per replicate       Mean body weight change of earthworms

             Test start       28 days       Per replicate       Mean ± SD       Sig.

             [g]       [g]       [g]       [%]       [g]       [%]       

Control       1       0.45       0.48       0.03       6.7       0.02 ± 0.03       3.91 ± 6.56       –

      2       0.46       0.45       -0.01       -2.2                     

      3       0.45       0.52       0.07       15.6                     

      4       0.46       0.43       -0.03       -6.5                     

      5       0.45       0.46       0.01       2.2                     

      6       0.45       0.48       0.03       6.7                     

      7       0.45       0.47       0.02       4.4                     

      8       0.45       0.47       0.02       4.4                     

62.5       1       0.45       0.47       0.02       4.4       0.02 ± 0.02       3.33 ± 4.62       No

      2       0.45       0.46       0.01       2.2                     

      3       0.45       0.44       -0.01       -2.2                     

      4       0.45       0.49       0.04       8.9                     

125       1       0.45       0.43       -0.02       -4.4       0.00 ± 0.02       0.55 ± 3.76       No

      2       0.45       0.46       0.01       2.2                     

      3       0.45       0.45       0.00       0.0                     

      4       0.45       0.47       0.02       4.4                     

250       1       0.45       0.49       0.04       8.9       0.03 ± 0.01       5.53 ± 2.25       No

      2       0.45       0.47       0.02       4.4                     

      3       0.45       0.47       0.02       4.4                     

      4       0.45       0.47       0.02       4.4                     

500       1       0.45       0.49       0.04       8.9       0.02 ± 0.02       4.45 ± 4.80       No

      2       0.45       0.48       0.03       6.7                     

      3       0.45       0.47       0.02       4.4                     

      4       0.45       0.44       -0.01       -2.2                     

1000       1       0.45       0.44       -0.01       -2.2       0.02 ± 0.02       3.90 ± 4.21       No

      2       0.45       0.48       0.03       6.7                     

      3       0.45       0.48       0.03       6.7                     

      4       0.45       0.47       0.02       4.4                     

SD = Standard deviation                            

Sig. = Statistical Significance (Dunnett’s Multiple Sequential t-test Procedure, α = 0.05) compared to control

Reproduction Rate (Number of Juveniles after 8 Weeks)

Application rate

[mg test item/kg SDW]       Replicate       Number of juveniles       Mean ± SD       CV               [%]       [%]

of control       Sig.

Control       1       121       115 ± 22.8       19.9       -       -

      2       95                            

      3       83                            

      4       117                            

      5       124                            

      6       159                            

      7       116                            

      8       102                            

62.5       1       133       125 ± 23.0       18.4       109       No

      2       101                            

      3       153                            

      4       112                            

125       1       123       133 ± 19.8       14.9       116       No

      2       124                            

      3       123                            

      4       163                            

250       1       131       123 ± 17.3       14.1       107       No

      2       141                            

      3       118                            

      4       101                            

500       1       97       86 ± 18.8       21.9       74.8       Yes

      2       97                            

      3       92                            

      4       58                            

1000       1       131       92 ± 27.5       29.9       80.0       Yes

      2       90                            

      3       70                            

      4       76                            

SD = Standard deviation       CV = Coefficient of variation      

Sig. = Statistical Significance (William’s Multiple Sequential t-test Procedure, α = 0.05) compared to the control

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study, neither evident earthworm mortalities (< 10%) nor pathological symptoms or changes in the behaviour of adult earthworms were observed in the control or all test item concentrations. There were no statistically significant differences in biomass in all test item concentrations compared to the control. After a further four weeks, the reproduction rate (average number of juveniles produced) was 115 in the control and ranged from 86 to 133 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction in the treatment rates 62.5 to 250 mg test item/kg SDW compared to the control. However, at the test item concentrations 500 and 1000 mg test item/kg SDW the earthworm reproduction was statistically significantly reduced.

All validity criteria recommended by the test guidelines were fulfilled.
Executive summary:

Effects of Sodium methyl oleoyl taurate on mortality, biomass and the reproductive potential of the earthworm species Eisenia fetida (Annelida, Lumbricidae) were determined according to OECD 222 (2016) from 2019-02-28 to 2019-08-16 with the definitive exposure phase from 2019-06-21 to 2019-08-16 at the test facility in 31157 Sarstedt, Germany.

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

Each concentration level and control were analysed via LC-MS/MS analysis on day 0 and day 55 using pooled samples of all replicates.

The measured concentrations of the pooled samples of replicates were within the range of 93 to 100 % of the nominal values on day 0, demonstrating the right preparation of the tested concentrations. The detailed analytical results are presented in part 6.1.4. The method was validated according to SANCO/3029/99 rev.4 (2000) prior to the start of exposure. Details of the analytical method are presented in part 14.

After 28 days of exposure in soil, neither evident earthworm mortalities (< 10%) nor pathological symptoms or changes in the behaviour of adult earthworms were observed in the control or all test item concentrations. There were no statistically significant differences in biomass in all test item concentrations compared to the control. After a further four weeks, the reproduction rate (average number of juveniles produced) was 115 in the control and ranged from 86 to 133 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction in the treatment rates 62.5 to 250 mg test item/kg SDW compared to the control. However, at the test item concentrations 500 and 1000 mg test item/kg SDW the earthworm reproduction was statistically significantly reduced.

All validity criteria recommended by the test guidelines were fulfilled.

Description of key information

Effects of Sodium methyl oleoyl taurate on mortality, biomass and the reproductive potential of the earthworm species Eisenia fetida (Annelida, Lumbricidae) were determined according to OECD 222 (2016) from 2019-02-28 to 2019-08-16 with the definitive exposure phase from 2019-06-21 to 2019-08-16 at the test facility in 31157 Sarstedt, Germany.

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

Each concentration level and control were analysed via LC-MS/MS analysis on day 0 and day 55 using pooled samples of all replicates.

The measured concentrations of the pooled samples of replicates were within the range of 93 to 100 % of the nominal values on day 0, demonstrating the right preparation of the tested concentrations. The detailed analytical results are presented in part 6.1.4. The method was validated according to SANCO/3029/99 rev.4 (2000) prior to the start of exposure. Details of the analytical method are presented in part 14.

After 28 days of exposure in soil, neither evident earthworm mortalities (< 10%) nor pathological symptoms or changes in the behaviour of adult earthworms were observed in the control or all test item concentrations. There were no statistically significant differences in biomass in all test item concentrations compared to the control. After a further four weeks, the reproduction rate (average number of juveniles produced) was 115 in the control and ranged from 86 to 133 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction in the treatment rates 62.5 to 250 mg test item/kg SDW compared to the control. However, at the test item concentrations 500 and 1000 mg test item/kg SDW the earthworm reproduction was statistically significantly reduced.

All validity criteria recommended by the test guidelines were fulfilled.

Key value for chemical safety assessment

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

Additional information