Ziram

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 March 1993 - 25 May 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Qualifier:

according to guideline

Guideline:

other: EEC Commission Directive 87/302/EEC

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

- The test substance was directly dispersed in basic soil substrate. 250, 450, 800, 1400 and 2500 mg of test material were each separetely washed into the main soil bulk of 2.5 kg (dry weight) artificial soil with 550 mL deionised water to give a moisture content of approximately 30% and mixed to give the test series

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earthworm
- Source: Blades Biological, Cowden, Kent, UK
- Weight at test initiation: 0.41 g

ACCLIMATION
- Acclimation period: 24 h
- Acclimation conditions: test organisms were kept in 100 L plastic containers, at 21°C and in darkness. As substrate a mixture of 50% by volume peat and 50% by volume animal manure was used. Once per week a mixture of animal manure and household vegetable waste was added as additional supplement.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

14 d

Test temperature:

21°C

pH:

6.2 +/- 0.1

Moisture:

28-30% of dry weight of basic substrate on day 0 and 26-27% on day 14

Details on test conditions:

TEST SYSTEM
- Test container: 1 L glass beakers covered by plastic films with ventilation holes
- Amount of soil or substrate: 500 g dry weight
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 4


SOURCE AND PROPERTIES OF SUBSTRATE
Artificial soil
- Industrial quartz sand 69 % w/w
- Kaolinite clay 20 % w/w
- Sphagnum moss peat 10 % w/w
- Calcium carbonate 1% % w/w (to bring pH within range 6 +/- 2)


OTHER TEST CONDITIONS
- Photoperiod: continuous
- Light intensity: 600 lux

EFFECT PARAMETERS MEASURED: mortality at 7 and 14 days

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.7
- Range finding study
- Test concentrations: 0.10, 1, 10, 100 and 1000 mg/kg
- Results used to determine the conditions for the definitive study: No mortalities occurred at any concentration but the 1000 mg/kg where 100%
mortality was reported

Nominal and measured concentrations:

Nominal concentrations: 100, 180, 320, 560 and 1000 mg a.i./kg

Reference substance (positive control):

yes

Remarks:

chloroacetamide

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

140 mg/kg soil dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: 95% CL: 140 - 150 mg/kg

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/kg soil dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Duration:

14 d

Dose descriptor:

LC0

Effect conc.:

100 mg/kg soil dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Duration:

14 d

Dose descriptor:

LC100

Effect conc.:

320 mg/kg soil dw

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Results with reference substance (positive control):

14d-LC50 for chloroacetamide was 19 mg/kg

Validity criteria fulfilled:

yes

Reference 2

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Jan - 25 Mar 1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Draft

Qualifier:

according to guideline

Guideline:

other: BBA-Guideline, part VI, 2-2

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministry for Environment and Traffic, Baden-Württenberg, Germany

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

- Method of application to soil surface (if used): The deionized water (control), the test substance solution and the toxic standard solution were applied, using an automatic laboratory spraying-cabin, simulating a field application. The spraying-cabin was calibrated before application by adjusting the spraying pressure, application speed and type of nozzle to provide an output of 600 L/ha.
- Controls: yes

Test organisms (species):

other: Mixed population of Eisenia fetida fetida and Eisenia fetida andrei

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earthworms
- Source: Kraut&Rüben (D-64367 Mühltal-Frankenhausen, Germany)
- Age at test initiation: At least 2 months old but not older than 1 year
- Weight at test initiation: between 300 and 600 mg


ACCLIMATION
- Acclimation period: 24 h
- Acclimation conditions: from the breeding medium and acclimated for 24 h in moistened artificial soil (without test substance).

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Test temperature:

18 - 22 °C

pH:

6.2 - 6.8

Moisture:

29.4 - 36.7 %

Details on test conditions:

TEST SYSTEM
- Test container (material, size): White plastic vessels (BELLAPLAST, 17 cmx12.5 cmx6 cm)
- Amount of soil or substrate: , 600 g dry artificial soil
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 4

SOURCE AND PROPERTIES OF SUBSTRATE
- Composition: - 10 % Sphagnum peat (close to pH 5.5 – 6.0, no visible plant remains, finely ground and air-dry);
- 20 % Kaolinite Clay (kaolinite content > 30 %)
- 69 % Industrial sand (fine sand dominant)
- ca. 1 % calcium carbonate (CaCO3) (pH was adjusted to 6.0 ± 0.5)

OTHER TEST CONDITIONS
- Photoperiod:Light dark cycle 18/8 h (long day conditions)

Nominal and measured concentrations:

Nominal: 4 (1-time application) and 20 (5-times application) kg formulated product/ha

Reference substance (positive control):

yes

Remarks:

carbendazim in the form of Derosal liquid

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

4.053 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: survival, reproduction and growth

Remarks on result:

other: NOEC calculated in mg a.s./kg soil dw retrospectively based on results of the study

No effects on survival, growth or reproduction of the test organisms was reported at the 4 kg formulated product/ha application rate

(4.053 mg a.s./kg dry weight soil)

Validity criteria fulfilled:

yes

Conclusions:

Validity criteria according to the ISO 11268-2 guideline are fulfilled, test results can be considered reliable.

Executive summary:

Materials and methods: The sub-lethal effects of ziram 76 % WG on the earthworm species Eisenia foetida were determined in a laboratory test using an artificial soil. The study was carried out following the BBA-Guideline, part VI 2-2, “Auswirkungen von Pflanzenschutzmitteln auf das Wachstum von Eisenia Fetida/Eisenia Andrei” and Draft International Standard ISO-Guideline 11268-2, “Effects of pollutants on earthworms (Eisenia foetida) – Part 2: Method for the determination of effects on reproduction. (Anonymus, 1993).. Application rates of 1´and 5´concentrations (i.e. 4 and 20 kg formulated product /ha) were tested.

Carbendazim was used as toxic reference item.

Results and discussion: After 4 weeks exposure to the test substrate a growth of the body weight was recorded in the 1´and 5´concentration compared to the control, whereas worms of the toxic standard group showed a reduction of the body weight, resulting significantly lower weight than that of the control. The food consumption of the test organisms in the toxic standard was distinctly reduced compared with the control.

No mortality was observed in the 1´and 5´concentration of ziram 76 % WG, in the control group and in the toxic standard group.

Concerning the number of offspring, a significant difference was calculated at the 5´concentration of ziram 76 % WG and the toxic standard compared with the control.

In the 1´concentration of ziram 76 % WG a significant increase of the number of juvenile earthworms could be calculated. Therefore, no impact of ziram 76 % WG at this concentration on the earthworm reproduction could be concluded.

No negative effects concerning the weight loss could be recorded even in the 5´concentration of ziram 76 % WG. Long-term effects could be observed only in the 5´concentration as decrease in the number of juvenile earthworms.

Reference 3

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 Apr - 03 Jun 2013

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:

adopted April 13, 2004

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

- Method of mixing test item into soil: A stock solution was prepared by weighing 91.0 mg of Ziram 76 WG. Deionised water was added to reach a final net weight of 1000 g. The resulting suspension contained a concentration of 0.091 mg Ziram 76 WG/g. A magnetic stirrer was used to obtain a homogenous dispersion of the test item. 95.5, 127.3, 169, 225.3 and 299.6 g of the stock solution was added to 2050 g dry artificial soil to prepare the target nominal concentrations. The control was left untreated. While mixing the artificial soil in a laboratory mixer for approximately 5 min the soil of each treatment group (including the control) was moistened with deionised water. Each group was treated in one batch (two in the control) which was then split into the replicates.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: earthworm
- Source: in-house culture (bred under standardised conditions in the laboratory in a breeding medium of cattle manure, peat, sand, calcium carbonate and straw, fed with cattle manure and stored at room temperature)
- Age at test initiation: adult worms (with clitellum), approximately 11 months old, age range between test individuals not differing by more than 4 weeks
- Weight at test initiation: weight range 324 to 600 mg

ACCLIMATION
- Acclimation period: 1 d
- Acclimation conditions: in artificial soil, under test conditions

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

55 d

Post exposure observation period:

no

Test temperature:

18 °C - 22 °C

pH:

Test start: 6.2
Test end (8 wk): 5.7-5.8

Moisture:

Test start: 29.9% to 30.8% (53.3% to 55.0% of maximum water holding capacity, WHC)
Test end (8 wk): 31.0% to 33.8% (55.3% to 60.3% of the maximum WHC)

Details on test conditions:

TEST SYSTEM
- Test container: Plastic boxes (18.3 cm x 13.6 cm x 6 cm, tapered towards the bottom, with a soil surface of approx. 16.5 cm x 11.5 cm = 189.75 cm²) with perforated transparent lids to enable exchange of air, minimise evaporation and to prevent the worms from escaping.
- Amount of soil or substrate: 650.9 g/box ((500 g dry weight plus 145.9 g deionised water plus 5 g food))
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate): Artificial soil was prepared according to OECD 222:
- 10% Sphagnum-peat, air-dried and finely ground (2 mm with no visible plant remains); (Floragard, Vertriebs GmbH für Gartenbau, 26138 Oldenburg, Germany)
- 20% Kaolin clay (Kaolinite content >30%) (Erbslöh, 65558 Lohrheim, Germany)
- 69.6% fine quartz-sand (F34) containing more than 50% by mass of particle size 0.05 mm to 0.2 mm; (Quarzwerke Frechen, Postfach 1780, 50207 Frechen, Germany)
- 0.4% Calcium carbonate (CaCO3) extra pure (Merck, 64293 Darmstadt, Germany) to adjust pH to 6.0 ± 0.5.
- The artificial soil was moistened to approximately half of the final water content 1 day before the application. The additional water required to achieve the final water content was added when applying the test item.
- Maximum water holding capacity (in % dry weigth): 56% of the dry weight
- Pretreatment of soil: no
- Stability and homogeneity of test material in the medium: refer to Section "details on preparation and application of test substrate"

FOOD
- Type: finely ground cattle manure
- Source: Cattle manure was supplied by H. Lautz, Ober-Ramstadt, Germany and prepared at the laboratory.
- Amount of food and application: 10 g food/kg dry soil was mixed into the artificial soil 1 day before the start of the study; 5 g/container was scattered on the soil surface at day 1 after application and was moistened with 5 g deionised water; 5 g/container (moistened with 2 - 3 g deionised water) was added each week for the first 4 weeks of the experiment, when the food of the previous week had almost been consumed. If the food was not quite fully consumed, the added amount of food was adjusted to replace the visually estimated consumption. Four weeks after application, the food was mixed into the substrate following removal of the adult worms.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light / 8 h dark
- Light intensity: within the range of 400 lux to 800 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
Mortality, body weight change, feeding activity, behavioural and morphological abnormalities and reproduction rate were determined. Assessment of adult worm mortality, behavioural effects and biomass development was carried out after 28 days exposure of adult worms in treated artificial soil. Reproduction rate (number of offspring) was assessed after additional 27 days (assessed 55 days after application).

TEST CONCENTRATIONS
- Range finding study: information not available

Nominal and measured concentrations:

Nominal: control, 4.24, 5.65, 7.50, 10.0 and 13.3 mg Ziram 76 WG/kg soil, equivalent to 3.22, 4.29, 5.70, 7.60 and 10.1 mg a.s./kg soil

Reference substance (positive control):

yes

Remarks:

Luxan Carbendazim 500 FC (Carbendazim, 500 g/L nominal)

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 10 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Duration:

55 d

Dose descriptor:

NOEC

Effect conc.:

>= 10 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 13 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

55 d

Dose descriptor:

NOEC

Effect conc.:

>= 13 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- Mortality at end of exposure period: no mortalities
- Total mass of adults at beginning of test:
- Changes in body weight of live adults (% of initial weight) at end of exposure period: Not statistically significantly different compared to the control up to and including the concentration of 10.0 mg Ziram 76 WG/kg soil. At the concentration of 13.3 mg Ziram 76 WG/kg soil the earthworms showed a statistically significant body weight increase (19.4 ± 5.1% increased, Williams t-test, α = 0.05, two-sided). A stronger weight increase is not considered to be an adverse effect, especially if the increase does not show a dose relation. Therefore, the NOAEC for body weight changes is proposed to be 13.3 mg Ziram 76 WG/kg soil (10.1 mg a.s./kg soil).
- No. of offspring produced: control: 274 - 377, treatments: 185 - 374
- No. of unhatched cocoons: not observed
- Morphological abnormalities: none mentioned in report
- Behavioural abnormalities: none observed
- Feeding activity: in all treatments comparable to the control
- Other biological observations: In all treatment groups, food was consumed. The results show that the turnover of biomass of those earthworms exposed to the different rates of the test item was comparable to the control.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 = 1.7 mg carbendazim/kg soil ( statistically significant effects on reproduction at a concentration of 1.30 mg carbendazim/kg soil and higher)

Reported statistics and error estimates:

Data for body weight changes and reproduction were normally distributed and homogeneous.

Table 1: Validity criteria for OECD 222 (2016)

Criterion from the guideline to be satisfied in the controls	Outcome	Validity criterion fulfilled
Adult mortality over the initial 4 weeks of the test is ≤ 10%.	Control mortality was 0%	yes
Each replicate (containing 10 adults) to have produced ≥ 30 juveniles by the end of the test.	The number of juvenile worms per replicate was 274 to 377	yes
The coefficient of variation of reproduction is ≤ 30%.	The coefficient of variation of reproduction 12.4%	yes

The reproduction was significantly reduced compared to the control at the concentration of 5.65 mg Ziram 76 WG/kg soil, which was not considered to be a treatment related effect since no significant reduction could be observed at all higher concentrations (Dunnett's t-test, α = 0.05, one-sided smaller). The NOEC for reproduction was determined to be 13.3 mg Ziram 76 WG/kg soil (10.1 mg a.s./kg soil).

Table 2: Reproduction of the earthworms after 8 weeks

Treatment group	container	significance	significance	% of control	significance
	#	per container	mean ± SD
control	1	285	322 ± 40	-	-
	2	377
	3	345
	4	371
	5	311
	6	283
	7	274
	8	332
4.24 mg Ziram 76 WG/kg	1	240	283 ± 34	87.9	n.s.
	2	282
	3	323
	4	288
5.65 mg Ziram 76 WG/kg	1	185	232 ± 71	72	*
	2	338
	3	209
	4	196
7.50 mg Ziram 76 WG/kg	1	224	282 ± 56	87.5	n.s.
	2	315
	3	247
	4	342
10.0 mg Ziram 76 WG/kg	1	282	317 ± 35	98.2	n.s.
	2	360
	3	330
	4	294
13.3 mg Ziram 76 WG/kg	1	336	312 ± 55	96.7	n.s.
	2	247
	3	290
	4	374

Validity criteria fulfilled:

yes

Description of key information

NOEC (55 d) ≥10.1 mg a.i./kg soil dw (nominal, Eisenia fetida, OECD 222)

LC50 (14 d) = 140 mg a.i./kg dry weight soil (arithm. mean meas., Eisenia fetida, OECD 207)

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:

140 mg/kg soil dw

Additional information

Two studies investigating the long-term and one study investigating the short-term effects of zinc bis dimethyldithiocarbamate (CAS No. 137-30-4) to earthworms are available.

In the key study (2013) the effects of Ziram 76 WG on the mortality, body weight and reproduction of adultEisenia fetidawere investigated according to the OECD Guideline No. 222 and ISO-Guideline 11268-2. The study was performed under GLP conditions. The test organisms in artificial soil were exposed for 55 d to the test item concentrations of 3.22, 4.29, 5.70, 7.60 and 10.1 mg a.i./kg soil and a control (artificial soil without test item). Carbendazim was used as a reference substance. The mortality was assessed after 28 d and the reproduction after 55 d. The study resulted into a NOEC (55 d) of=10.1 mg a.i./kg soil dw and a NOEC (28 d) of≥10.1 mg a.i./kg soil dw (both nominal) for reproduction and mortality, respectively.

The long-term effects of Ziram 76 WG in earthworms were investigated in a further study (1999), which was performed according to the Draft ISO-Guideline 11268-2 (1993), BBA Guideline, part VI, 2-2 (1995) and under GLP conditions. The test substance was applied as a spray solution on the soil (1-time application and 5-time application corresponding to 4 and 20 kg formulated product/ha, respectively). Effects on survival and biomass were observed after 28 days of exposure, followed by an additional 28-days period where reproduction-related effects were assessed. No mortality was reported at any concentration tested after 28 d. Adverse effects on reproduction (based on number of offspring) were observed only at the highest concentration of 20 kg formulation/ha. On this basis, the NOEC (56 d) was retrospectively calculated as 4.053 mg a.i./kg soil dw (4 kg formulation/ha) based on results of the study. The data of this study have been used as supporting information, since the test design of the chosen key study was more appropriate to acquire a reliable dose response relationship.

The short-term toxicity of zinc bis dimethyldithiocarbamate (CAS No. 137-30-4) to earthworms was investigated in a study (1993) according to the OECD Guideline No. 207 (1984) and under GLP conditions. Earthworms were exposed for 14 days to the test material in artificial soil at concentrations ranging from 100 to 1000 mg a.i./kg dry weight soil. The resulting 14 d-LC50 for mortality was determined to be 140 mg a.i./kg dry weight soil (arithm. mean meas.). The data of this study were used as supporting information.