mancozeb (ISO); manganese ethylenebis(dithiocarbamate) (polymeric) complex with zinc salt  

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:

1999-08-27 to 199-10-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

not applicable

Details on preparation and application of test substrate:

A stock solution equivalent to 97.9192 g/L was prepared by diluting 97.9190 g formulation of Dithane M-45 with deionized water to a total volume of 1000 mL, obtaining a concentration equivalent to 48 g a.i./ha applied in an application volume of 600 L/ha. Thereafter, the stock was serially diluted by taking aliquots of 500 mL and diluting it with deionized water to a total volume of 1000 mL. The following solution concentrations were thus obtained: 40, 20, 10 and 5 g a.i./L, which corresponds to 24, 12, 6, and 3 kg a.i./ha, respectively. After the earthworms had burrowed beneath the soil surface, the stock solutions were applied using an SL Conformal Spray System (model RC-10E, Springborn Laboratories). Prior to the application of the treatments, the precision sprayer (SL Conformal Spray System, Model RC-10E, Springborn Laboratories) was calibrated with deionized water to deliver a target volume of 6 mg of spray solution per ern" (600 Uha) by weighing the amount of water delivered during a set spray interval. After calibration, the sprayer delivered 6.05 mg/cm2. Before application, the spray pattern was visually checked for uniformity. Before, between, and after applications (except after the control), the sprayer was cleaned with hot water and detergent, an acetone-water solution, and finally rinsed with deionized water.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: redworm
- Source: cultured in-house (originally received from received from the Biologische Bundesanstalt (BBA), Braunschweig, Germany, on September 20, 1996)
- Age at test initiation: 7 to 8 months old adult worms
- Weight at test initiation: mean fresh weight of 488 ± 32 mg per earthworm (450 to 550 mg)

ACCLIMATION
- Acclimation period: seven days prior to test initiation
- Acclimation conditions: placed on artificial soil for acclimation and fed with cow manure ad libitum
- Health during acclimation: not specified

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Test temperature:

20 ± 2°C

Details on test conditions:

TEST SYSTEM
- Test container (material, size): plastic, 2 L (dimensions 17 x 12.5 x 11.5 cm, approximately 212.5 cm^2 surface on top), with a perforated plastic lid allowing gas exchange but preventing worms from escaping
- Amount of soil or substrate: a total amount of 550 g dry artificial soil (659.51 g moist soil of 19.91% moisture), 5.5 g of dry cow manure and 40.27 g of deionized
water was mixed for each replicate.
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 4

PROPERTIES OF SUBSTRATE
- 70 % Industrial sand (at least 50% of particles are 50 to 200 11m)
- 20 % Kaolin clay (kaolinite > 30%)
- 10 % Sphagnum peat (no visible plant remains; finely ground)

OTHER TEST CONDITIONS
- Photoperiod: 16L: 80
- Light intensity: 400-800 lux by fluorescent lights

EFFECT PARAMETERS MEASURED: mortality and biomass (growth)

TEST CONCENTRATIONS
- Range finding study: performed
- Test concentrations: 6, 12, 24 and 48 kg a.i./ha
- Results used to determine the conditions for the definitive study: yes

Nominal and measured concentrations:

Nominal: 0 (control), 3, 6, 12, 24 and 48 kg a.i./ha (equivalent under the conditions of this study to 0, 10, 20, 40, 80 and 161 mg a.i./kg dry soil, respectively)

Reference substance (positive control):

yes

Remarks:

Reference substance: Benlate

Key result

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

10 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

not specified

Remarks on result:

other: derived by Rapporteur Member States in the course of evaluation according to Regulation (EU) N0 1107/2009.

Key result

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

161 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth

Key result

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

161 mg/kg soil ww

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Key result

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

20 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Details on results:

There were no statistically significant differences between the control and the treatment levels in mortality or growth (biomass). Earthworm reproduction (number of offspring) was significantly affected in the 12, 24 and 48 kg a.i./ha treatments (equivalent to nominal concentrations of 40, 80 and 161 mg a.i./kg dry soil, or 3x, 6x and 12x the application rate of 4.0 kg a.i./ha). The chronic no observed effect concentration (NOEC) for earthworms was 6.0 kg a.i./ha (20 mg a.i./kg).

Results with reference substance (positive control):

The reference test substance, benomyl, exhibited a 28 day LC50 of 12.9 mg a.i./kg soil and a reproduction and growth NOEC of 5 and 1 mg a.i./kg soil, respectively, demonstrating that the test protocol was adequate to detect sublethal toxicity. The LOEC was estimated to be 10 and 15 mg benomyl/kg dry for the same parameters.

Reported statistics and error estimates:

Mortality data were not tested statistically since no mortality was observed. Adult earthworm growth and reproduction data (fresh weight and number of offspring) were analysed for normality using the Shapiro-Wilk's Test. If the data met the assumption of normality, they were tested for statistically significant differences between treatments using ANOVA followed by Tukey HSD (Sokal and Rohlf, 1995). If the assumption of normality was not met, the Kruskal-Wallis ANOVA test was used (Sokal and Rohlf, 1995). Significance level was a = 0.05. The computer program used to perform the statistical analyses was "Statistica for Windows" Version 5.0, by StatSoft Inc., Tulsa OK, USA.

Validity criteria fulfilled:

yes

Conclusions:

In a GLP-compliant study according to ISO Guideline 11268-2 with Eisenia fetida the 28-day LC50 was determined to be > 161 mg a.i./kg. The 28 day no-observed-effect-concentration (NOEC) for mortality and growth was 161 mg a.i./kg. The 56-day NOEC for reproduction was 20 mg a.i./kg based on reduced number of offspring per adult earthworm. The NOECcorr considered for the Risk Assessement is 10 mg a.s./kg dw soil.

Executive summary:

To assess the long-term toxicity of the preparation Dithane M-45 (81.7% a.i.) to earthworms a study Eisenia fetida according to ISO Guideline 11268-2 and GLP principles was carried out. The test item was administered to earthworms (Eisenia foetida) during an 8 week (56 day) chronic toxicity and reproduction study. In the chronic (56 day) earthworm toxicity test, four replicates (10 worms per replicate) were exposed to surface applications of the test item at treatment levels of 0 (control), 3, 6, 12, 24 and 48 kg a.i./ha (equivalent under the conditions of this study to 0, 10, 20, 40, 80 and 161 mg a.i./kg dry soil, respectively). A positive reference substance, Benlate (benomyl, 50% a.i.), was also tested to ensure that the study procedures could adequately detect sublethal toxicity. The mean biomass loading was 0.18 g/20 g soil, at test initiation and did not exceed the maximum loading rate of 1.0 g biomass/20 g soil as specified in the test guidelines. After four weeks of exposure, mortality and growth were evaluated. Cocoons containing the next generation of worms were left in the test exposure beakers and were allowed to hatch over the next 28 days in order to assess effects on reproduction. On Day 56, reproduction (the number of juvenile worms produced) was assessed. The test soil was artificial soil prepared according to OECD Guideline 207 with a pH of 6.2 at test initiation. Photoperiod was 16 hours light: 8 hours darkness. Other environmental parameters including temperature (19 - 21.0 °C), pH (mean Day 0, 6.2; mean Day 56, 6.8), light intensity (487 - 602 lux) and soil moisture as a percent of water holding capacity (60-78%) were within acceptable limits. As a result, the 28-day LC50 for the earthworm was found to be > 161 mg a.i./kg, the highest concentration tested. The 28 day no-observed-effect-concentration (NOEC) for mortality and growth was 161 mg a.i./kg. The 56-day NOEC for reproduction was 20 mg a.i./kg based on reduced number of offspring per adult earthworm. In conclusion, treatments at up to 48 kg a.i./ha did not significantly affect mortality and growth of adult Eisenia fetida. Earthworm reproduction (number of offspring) was not significantly affected at treatment levels of 3 and 6 kg a.i./ha (equivalent to nominal concentrations of 10 and 20 mg a.i./ kg dry soil). However, offspring production was significantly affected in the 12, 24 and 48 kg a.i./ha treatments (equivalent to nominal concentrations of 40,80 and 161 mg a.i./ kg dry soil). A corrected NOEC has been agreed with Rapporteur Member States in accordance with Regulation (EU) No 1107/2009: NOECcorr = 10 mg a.s./kg dw soil. This key value is considered suitable for the Risk Assessment.

Description of key information

Short-term toxicity to soil macroorganisms except arthropods

In accordance with REACH Annex XI a short-term study does not need to be conducted because an appropriate long-term toxicity study on terrestrial organisms is available. Additional data on short-term toxicity is scientifically not necessary; therefore, this endpoint was waived.

Long-term toxicity to soil macroorganisms except arthropods

In a GLP-compliant study according to ISO Guideline 11268-2 with Eisenia fetida the 28-day LC50 was determined to be > 161 mg a.s./kg. The 28 day no-observed-effect-concentration (NOEC) for mortality and growth was 161 mg a.s./kg. The 56-day NOEC for reproduction was 20 mg a.s./kg based on reduced number of offspring per adult earthworm. The NOECcorr considered for the Risk Assessement is 10 mg a.s./kg dw soil.

Key value for chemical safety assessment

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

10 mg/kg soil dw

Additional information

Data of two guideline studies on the long-term toxicity of Mancozep preparations are available and presented below.

Key study

A chronic toxicity and reproduction test exposing the earthworm Eisenia foetida to Dithane M-45 in OECD artificial soil (Nienstedt 1999 (Doc. No. 833-014, cross reference to Risk Assessment Report according to Regulation (EU) No 1107/2009: 10.6.1.2/01))

To assess the long-term toxicity of the preparation Dithane M-45 (81.7% a.i.) to earthworms a study according to ISO Guideline 11268-2 and GLP principles was carried out. The test item was administered to Eisenia foetida during an 8 week (56 day) chronic toxicity and reproduction study. Four replicates (10 worms per replicate) were exposed to surface applications of the test item at treatment levels of 0 (control), 3, 6, 12, 24 and 48 kg a.s./ha (equivalent under the conditions of this study to 0, 10, 20, 40, 80 and 161 mg a.s./kg dry soil, respectively). A positive reference substance, Benlate (benomyl, 50% a.i.), was also tested to ensure that the study procedures adequately detect sublethal toxicity. The mean biomass loading was 0.18 g/20 g soil, at test initiation and did not exceed the maximum loading rate of 1.0 g biomass/20 g soil as specified in the test guideline. After four weeks of exposure, mortality and growth were evaluated. Cocoons containing the next generation of worms were left in the test exposure beakers and were allowed to hatch over the next 28 days in order to assess effects on reproduction. On Day 56, reproduction (the number of juvenile worms produced) was assessed. The test soil was artificial soil prepared according to OECD Guideline 207 with a pH of 6.2 at test initiation. Photoperiod was 16 hours light: 8 hours darkness. Other environmental parameters including temperature (19 - 21.0 °C), pH (mean Day 0, 6.2; mean Day 56, 6.8), light intensity (487 - 602 lux) and soil moisture as a percent of water holding capacity (60-78%) were within acceptable limits. As a result, the 28-day LC50 for the earthworm was found to be > 161 mg a.s./kg, the highest concentration tested. The 28 day NOEC for mortality and growth was 161 mg a.s./kg. The 56-day NOEC for reproduction was 20 mg a.i./kg based on reduced number of offspring per adult earthworm. In conclusion, treatments at up to 48 kg a.i./ha did not significantly affect mortality and growth of adult Eisenia fetida. Earthworm reproduction (number of offspring) was not significantly affected at treatment levels of 3 and 6 kg a.s./ha (equivalent to nominal concentrations of 10 and 20 mg a.s./ kg dry soil). However, offspring production was significantly affected in the 12, 24 and 48 kg a.s./ha treatments (equivalent to nominal concentrations of 40,80 and 161 mg a.s./ kg dry soil). A corrected NOEC has been agreed with Rapporteur Member States in accordance with Regulation (EU) No 1107/2009: NOECcorr = 10 mg a.s./kg dw soil. This key value is considered suitable for the Risk Assessment.

Supporting information

Mancozeb: Reproduction toxicity to the earthworm Eisenia fetida in artificial soil with 5% peat  (Scheffczyk 2016 (Doc. No. 833-021, cross reference to Risk Assessment Report according to Regulation (EU) No 1107/2009: 10.4.1.1/01))

One additional study on the long-term toxicity of the Mancozeb preparation Dithane 45 (82.6% Mancozeb) to Eisenia fetida is available and used as supporting information. The earthworms were tested in an artificial soil. The composition of this artificial soil was based on OECD Guideline No. 222 (2004). 48 hours prior to test start, the artificial soil was prepared and deionised water was added to the dry components to adjust the water content to approximately 40-60 % of its maximum water holding capacity (WHC). The worms were acclimatised in a separate batch of the untreated artificial substrate for approximately five days before test start. The test concentrations were prepared by dispersing an exactly weighed amount of the test material in deionised water to make a stock solution. This stock solution was used to produce a range of dosage solutions for each concentration. An appropriate volume of the stock or respective dosage solution was applied to the artificial soil to achieve the required test item soil concentrations. The test item was incorporated into the soil by thoroughly mixing in the respective dosage solution for 5 minutes. Finally, the treated soil was placed into the test vessels. Per test vessel an amount corresponding to 500 g dw (dry weight) was used. 10 animals were put onto the soil surface of each test container. After the start of the experiment, food was first provided one day after application of the test item and introduction of the adult earthworms. The feeding activity was recorded for each test container at day 7, 14 and 21. 5 g dried food material was put on the soil surface of each test container. The food was moistened with 10 mL deionised water per test container. During the study water losses were compensated by addition of deionised water. After four weeks, the adult worms were removed from the test vessels, and mortality and the body weight of the surviving worms were determined. Morphological and behavioral changes of surviving adult earthworms were recorded as well. After all of the adult worms had been removed, the soil in each vessel was mixed with 5 g dried food material and moistened with 8 - 10 mL deionized water. Four weeks later, the number of surviving juveniles and any morphological and behavioral changes were recorded. As a result, 5 % mortality was observed in the control. In the eight tested concentrations of Mancozeb the mortality ranged from 0 to 15 %; there was no dose response observed. Fisher’s Exact Test (1-sided, p ≤ 0.05) showed no significant differences concerning mortality between the control and all test item concentrations. Statistical analysis showed no significant difference (Dunnett’s test, 2-sided; p ≤ 0.05) concerning biomass development of individual adults over 28 days between the control and all concentrations of the test item. Statistical analysis (Williams t-test; 1-sided, p ≤ 0.05) showed a significant difference concerning the number of juveniles between the control and the concentrations of 100, 178 and 316 mg a.s./kg soil (dw). All other concentrations tested did not differ statistically significantly from the control. Feeding activity was reduced in the control and most of the test item groups in some of the replicates at some of the observation times. There was no dose response of this effect. As a result, the EC10 and NOEC was found to be 59.4 and 56.2 mg a.s./kg soil dw, respectively. These values were corrected by Rapporteur Member States in accordance with Regulation (EU) No 1107/2009. The EC10corr and NOECcorr was determined  to be 28.1 and 29.7 mg a.s./kg soil dw, respectively.

This study is not documented in an individual IUCLID Endpoint Study Record. For details on test conditions and findings please refer to the disseminated EFSA Conclusion on the active substance Mancozep (Peer review of the pesticide risk assessment of the active substance mancozeb; European Food Safety Authority (EFSA), approved 12 June 2019, doi:10.2903/ j.efsa.2019.5755).

In accordance with Regulation (EU) No 1107/2009 both the key study as well as the supporting study were peer reviewed by Rapporteur Member States and have been proven to be valid  by the EFSA.

Conclusion

In a GLP-compliant study according to ISO Guideline 11268-2 with Eisenia fetida by Nienstedt (1999) the 28-day LC50 was determined to be > 161 mg a.s./kg. The 28 day NOEC for mortality and growth was 161 mg a.s./kg. The 56-day NOEC for reproduction was 20 mg a.s./kg based on reduced number of offspring per adult earthworm. The NOECcorr considered for the Risk Assessement is 10 mg a.s./kg dw soil. This result is supported by a study by Scheffczyk (2016) according to OECD Guideline No. 222 with Eisenia fetida: the EC10 and NOEC was found to be 59.4 and 56.2 mg a.s./kg soil dw, respectively. The EC10corr and NOECcorr was determined  to be 28.1 and 29.7 mg a.s./kg soil dw, respectively. Following a worst case approach the study of Nienstedt (1999) is considered as key information and the NOECcorr of 10 mg a.s./kg dw soil is taken into account for the risk assessment. The provided information is regarded reliable and sufficient to cover the data requirements according to REACH Annex X, section 9.4.4.