Thiophanate-methyl

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-02-07 to 2017-06-06

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:

July 29, 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 11268-2 (Effects of Pollutants on Earthworms. 2. Determination of Effects on Reproduction)

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

yes

Remarks:

Acetone

Details on preparation and application of test substrate:

The composition of this artificial soil was based on OECD Guideline No. 222 (2016) (the percentages refer to the dry weight):
- 10 % Sphagnum peat (no visible plant remains; finely ground and air-dried)
- 20 % Kaolin-Clay (kaolinite content > 30 %)
- 69 – 70 % Quartz sand (fine sand content with particles between 50 and 200 microns higher than 50 %)
- 0.3 - 1 % Calcium carbonate (CaCO3).
The pH value was adjusted to 6 ± 0.5 using calcium carbonate. The moisture content was adjusted to 40-60 % of WHCmax using deionised water. The water content of the test substrate was checked weekly by reweighing the test vessels and losses of water were compensated.

The stock solution was mixed with a magnet stirrer for 5 minutes. The dosage solutions were stirred for 2 minutes. 55 mL of the respective solutions were spiked on 210 g quartz sand (2 x 55 mL acetone for the solvent control). After evaporation of the solvent under a fume hood, the coated quartz sand was incorporated into 1890 g artificial soil (total amount of 2100 g) to prepare the different test item soil concentrations. Water was added to adjust the final soil moisture of 40 to 60 % of WHCmax. The prepared soil was mixed for 5 min.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earthworms
- Source: Co. Landenberger (D-72355 Schömberg)
- Age at test initiation: at least two months, but not more than one year old
- Weight at test initiation: 300 – 600 mg

ACCLIMATION
- Acclimation period: 24 hours
- Acclimation conditions: same as test
- Health during acclimation: no mortality observed

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Test temperature:

20 ± 2 °C

pH:

6 ± 0.5 °C

Moisture:

40 - 60 % of WHCmax

Details on test conditions:

TEST SYSTEM
- Test container (material, size): Bellaplast containers consisting of inert (non-toxic) plastic (Polystyrol); base area of 11 x 15.5 cm (= 170.5 cm2) and a height of 6 cm
- Amount of soil or substrate: 500 g dw
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 1
- No. of replicates per vehicle control: 2

OTHER TEST CONDITIONS
- Photoperiod: 16 h light: 8 h dark
- Light intensity: 400 - 800 Iux

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Test concentrations: 0.085, 0.152, 0.274, 0.494, 0.889, 1.60, 2.88 and 5.18 mg/kg soil (dw)

Nominal and measured concentrations:

Nominal: 0.085, 0.152, 0.274, 0.494, 0.889, 1.60, 2.88 and 5.18 mg/kg soil (dw)
Measured: not determined

Reference substance (positive control):

yes

Remarks:

Boric acid

Key result

Duration:

56 d

Dose descriptor:

EC50

Effect conc.:

1.91 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95 % confidence limits = 1.39 - 2.63 mg/kg soil (dw)

Key result

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

1.6 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

LOEC

Effect conc.:

2.88 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.:

1.53 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95 % confidence limits = 1.11 - 2.12 mg/kg soil (dw)

Key result

Duration:

56 d

Dose descriptor:

EC10

Effect conc.:

1.36 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95 % confidence limits = 0.88 - 2.11 mg/kg soil (dw)

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

5.18 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

2.88 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

LC50

Effect conc.:

> 5.18 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

5.18 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Biomass

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

2.88 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Biomass

Details on results:

- Mortality at end of exposure period: < 50 % in all concentrations
- Total mass of adults at beginning of test: 370.6 ± 28.0 mg to 379.0 ± 29.3 mg
- Changes in body weight of live adults (% of initial weight) at end of exposure period: between -37.4 - 64.8 %
- No. of offspring produced: 351.8 and 378.5 in the control and solvent control; uveniles in the test item treatment groups ranged from 0.0 to 428.0.
- Morphological abnormalities: none
- Behavioural abnormalities: none

Results with reference substance (positive control):

In a separate GLP study, the EC50 value was calculated by Probit analysis using Linear Max. Likelihood Regression as 325.4 mg boric acid/kg soil (dw) (% confidence limits = 314.6 - 349.7 mg boric acid/kg artificial soil (dw)). The estimated EC50 value in this study is slightly lower than aspired by the guideline. However, since the calculated EC50 is lower by factor of 1.3 than the expected range of 400 - 600 mg boric acid/ kg soil (dw) acceptable sensitivity of the test system is assumed.

Reported statistics and error estimates:

Statistical evaluation:
Mortality: Fisher's Exact Test with Bonferroni Correction (1-sided, α = 0.05).
Biomass change: Data were checked for normality by R/s test procedure and for homogeneity by Levene's test. Afterwards, treatment means were compared by Dunnett's test (α = 0.05, two-sided) and tested for statistically significant differences compared to the solvent control.
Reproduction: Data were checked for normality by R/s test procedure and for homogeneity by Cochran's test. Treatment means were compared by ANOVA followed by William's test (α = 0.05, one-sided) and tested for statistically significant differences compared to the solvent control (Sachs 1982).
The statistical software package ToxRat Professional 3.2.1 was used for these calculations.

Table 1: Number of dead earthworms after 28 days (n = 80 for solvent control; n = 40 for control and the concentrations). Data are given as absolute values and in percent of the total number (mortality).

Concentration [mg Thiophanate-methyl/ kg soil (dw)]	Number of dead adult worms	Mortality [%]
	Day 28	Day 28
Control	0	0.0
Solvent Control	1	1.25
0.085	0	0.0
0.152	1	2.5
0.274	0	0.0
0.494	1	2.5
0.889	0	0.0
1.60	0	0.0
2.88	1	2.5
5.18	18	45.0*

*Significantly different to solvent control (Fisher exact test, α = 0.05, one-sided)

 

Table 2: Average live weight (mg) per surviving adult earthworm at the beginning of the test and after 28 days (n = 80 for solvent control; n = 40 for control and the concentrations). Data are given as absolute value and in mean percent of the initial weight change per replicate.

Concentration [mg Thiophanate-methyl/ kg soil (dw)]	Biomass (mg fresh weight)		Mean change in % of the initial weight per replicate
	Day 0	Day 28	Day 28
Control	379.0 ± 29.3	610.6 ± 28.0	61.6
Solvent Control	376.6 ± 28.0	564.4 ± 30.8	50 5
0.085	377.8 ± 22.2	556.8 ± 34.1	47.4
0.152	373.0 ± 23.9	612.0 ± 20.7	64.8
0.274	370.6 ± 28.0	592.7 ± 47.7	60.0
0.494	375.3 ± 21.1	601.8 ± 37.4	61.0
0.889	375.7 ± 18.8	598.5 ± 41.3	59.2
1.60	371.3 ± 22.1	615.2 ± 27.8	66.1
2.88	373.9 ± 20.6	532.7 ± 16.8	42.6
5.18	377.5 ± 16.2	238.1 ± 97.3	-37.4*

* Significantly different to solvent control (Dunnett test, α = 0.05, two-sided)

 

Table 3: Reproduction after 56 days. Data are given as mean number of juveniles ± standard deviation and as % of the solvent control (n = 8 for solvent control; n = 4 for treatment and deionised water control).

Concentration [mg Thiophanate-methyl/ kg soil (dw)]	Number of Juveniles per Treatment (mean ± sd)	Number of Juveniles [% of Solvent Control]
Control	351.8 ± 40.6	92.9
Solvent Control	378.5 ± 84.3	100.0
0.085	411.3 ± 116.0	108.7
0.152	388.3 ± 71.7	102.6
0.274	354.0 ± 73.0	93.5
0.494	428.0 ± 125.7	113.1
0.889	346.8 ± 109.9	91.6
1.60	285.0 ± 130.8	75.3
2.88	21.0 ± 12.4	5.5 *
5.18	0.0 ± 0.0	0.0 *

*significantly different to solvent control (Williams t-test, α = 0.05, one-sided)

 

Validity Criteria

Mortality of the adult test animals in the solvent control (required: equal or less than 10 %): 1.25 %

Mean number of juveniles per replicate in the solvent control (required: > 30): (± standard 378.5 ± 84.3 deviation)

The coefficient of variation for the number of juveniles in the solvent (required: < 30 %): 22.3 % control

The data provide evidence that the quality criteria have been fulfilled.

Validity criteria fulfilled:

yes

Conclusions:

In a test according to OECD 222, the following endpoints were determined:
NOEC Mortality = 2.88 mg/kg soil (dw)
LOEC Mortality= 5.18 mg/kg soil (dw)
LC50 > 5.18 mg/kg soil (dw)
NOEC Reproduction = 1.60 mg/kg soil (dw)
LOEC Reproduction = 2.88 mg/kg soil (dw)
EC10 = 1.36 mg/kg soil (dw)
(95 % confidence limits = 0.88 - 2.11 mg/kg soil (dw)).
EC20 = 1.53 mg/kg soil (dw)
(95 % confidence limits = 1.11 -2.12 mg/kg soil (dw)).
EC50 = 1.91 mg/kg soil (dw)
(95 % confidence limits = 1.39 - 2.63 mg/kg soil (dw)).
NOEC Biomass = 2.88 mg/kg soil (dw)
LOEC Biomass = 5.18 mg/kg soil (dw).

Executive summary:

Reproduction toxicity of the test item to the earthworm Eienia fetida in artificial soil with 10% peat was assessed in a study according to OECD Test Guideline 222 and ISO 11268-2 under GLP conditions. The mortality and biomass were determined after 28 days while the reproduction was determined after 56 days. The following endpoints were determined: NOEC/LOEC_Mortality NOEC/LOEC_Biomass, NOEC/LOEC_Reproduction, and EC_X values for reproduction. Statistical analysis was evaluated by Probit analysis using maximum likelihood regression, Fisher's Exact Test with Bonferroni Correction (1-sided, α = 0.05); ANOVA followed by Williams t-test for reproduction (1-sided, α = 0.05); Dunnetts t-test for biomass (2-sided, α = 0.05). All validity criteria were met. The following endpoints were observed.

Mortality

0 % mortality was observed in the control, 1.25 % in the solvent control and 0 - 45.0 % mortality in all concentrations of the test item tested.

Fisher's Exact Test (1-sided, α = 0.05) showed a significant difference concerning mortality between the solvent control and the highest test item concentration, e.g. 5.18 mg/kg soil (dw).

Therefore, NOEC_Mortality was determined to be 2.88 mg/kg soil (dw). Accordingly the LOEC_Mortality was regarded to be 5.18 mg/kg soil (dw).

Since mortality was < 50 % in all concentrations of the test item tested no LC₅₀ value could be calculated but may be regarded to be > 5.18 mg/kg soil (dw).

Biomass change

Statistical analysis showed a significant difference (Dunnetts test, 2-sided; α = 0.05) concerning biomass development of individual adults over 28 days between the solvent control and the highest concentration of the test item tested.

Therefore, the NOEC_Biomass was determined to be 2.88 mg/kg soil (dw). The LOEC_Biomass is regarded to be 5.18 mg/kg soil (dw).

Reproduction

The mean number of juveniles was 351.8 and 378.5 in the control and solvent control, respectively. The mean number of juveniles in the test item treatment groups ranged from 0.0 to 428.0.

Statistical analysis (Williams test; 1-sided, α= 0.05) showed a significant difference concerning the number of juveniles between the solvent control and the two highest concentrations of the test item tested, e.g. 2.88 and 5.18 mg/kg soil (dw).

Therefore, the NOEC_Reproduction was determined to be 1.60 mg/kg soil (dw). The LOEC_Reproduction is regarded to be 2.88 mg/kg soil (dw).

The EC_X values were calculated using Probit analysis applying linear maximum likelihood regression.

The EC₁₀ was calculated to be 1.36 mg/kg soil (dw) (95 % confidence limits = 0.88 - 2.11 mg/kg soil (dw)).

The EC₂₀ was calculated to be 1.53 mg/kg soil (dw) (95 % confidence limits = 1.11 -2.12 mg/kg soil (dw)).

The EC₅₀ was calculated to be 1.91 mg/kg soil (dw) (95 % confidence limits = 1.39 - 2.63 mg/kg soil (dw)).

Description of key information

In a test according to OECD 222, the following endpoints were determined:

NOEC_(Mortality) = 2.88 mg/kg soil (dw)

LOEC_(Mortality) = 5.18 mg/kg soil (dw)

LC_{50 (Mortality)} > 5.18 mg/kg soil (dw)

NOEC_Reproduction = 1.60 mg/kg soil (dw)

LOEC_Reproduction = 2.88 mg/kg soil (dw)

EC_{10(Reproduction)} = 1.36 mg/kg soil (dw) (95 % confidence limits = 0.88 - 2.11 mg/kg soil (dw)).

EC_{20(Reproduction)} = 1.53 mg/kg soil (dw) (95 % confidence limits = 1.11 -2.12 mg/kg soil (dw)).

EC_{50(Reproduction)} = 1.91 mg/kg soil (dw) (95 % confidence limits = 1.39 - 2.63 mg/kg soil (dw)).

NOEC_Biomass = 2.88 mg/kg soil (dw)

LOEC_Biomass = 5.18 mg/kg soil (dw).

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for soil macroorganisms:

1.6 mg/kg soil dw

Additional information

Reproduction toxicity of the test item to the earthworm Eienia fetida in artificial soil with 10% peat was assessed in a study according to OECD Test Guideline 222 and ISO 11268-2 under GLP conditions. The mortality and biomass were determined after 28 days while the reproduction was determined after 56 days. The following endpoints were determined: NOEC/LOEC_Mortality NOEC/LOEC_Biomass, NOEC/LOEC_Reproduction, and EC_X values for reproduction. Statistical analysis was evaluated by Probit analysis using maximum likelihood regression, Fisher's Exact Test with Bonferroni Correction (1-sided, α = 0.05); ANOVA followed by Williams t-test for reproduction (1-sided, α = 0.05); Dunnetts t-test for biomass (2-sided, α = 0.05). All validity criteria were met. The following endpoints were observed.

Mortality

0 % mortality was observed in the control, 1.25 % in the solvent control and 0 - 45.0 % mortality in all concentrations of the test item tested.

Fisher's Exact Test (1-sided, α = 0.05) showed a significant difference concerning mortality between the solvent control and the highest test item concentration, e.g. 5.18 mg/kg soil (dw).

Therefore, NOEC_Mortality was determined to be 2.88 mg/kg soil (dw). Accordingly the LOEC_Mortality was regarded to be 5.18 mg/kg soil (dw).

Since mortality was < 50 % in all concentrations of the test item tested no LC₅₀ value could be calculated but may be regarded to be > 5.18 mg/kg soil (dw).

Biomass change

Statistical analysis showed a significant difference (Dunnetts test, 2-sided; α = 0.05) concerning biomass development of individual adults over 28 days between the solvent control and the highest concentration of the test item tested.

Therefore, the NOEC_Biomass was determined to be 2.88 mg/kg soil (dw). The LOEC_Biomass is regarded to be 5.18 mg/kg soil (dw).

Reproduction

The mean number of juveniles was 351.8 and 378.5 in the control and solvent control, respectively. The mean number of juveniles in the test item treatment groups ranged from 0.0 to 428.0.

Statistical analysis (Williams test; 1-sided, α= 0.05) showed a significant difference concerning the number of juveniles between the solvent control and the two highest concentrations of the test item tested, e.g. 2.88 and 5.18 mg/kg soil (dw).

Therefore, the NOEC_Reproduction was determined to be 1.60 mg/kg soil (dw). The LOEC_Reproduction is regarded to be 2.88 mg/kg soil (dw).

The EC_X values were calculated using Probit analysis applying linear maximum likelihood regression.

The EC₁₀ was calculated to be 1.36 mg/kg soil (dw) (95 % confidence limits = 0.88 - 2.11 mg/kg soil (dw)).

The EC₂₀ was calculated to be 1.53 mg/kg soil (dw) (95 % confidence limits = 1.11 -2.12 mg/kg soil (dw)).

The EC₅₀ was calculated to be 1.91 mg/kg soil (dw) (95 % confidence limits = 1.39 - 2.63 mg/kg soil (dw)).