polymeric zinc propylenebis(dithiocarbamate);propineb (ISO)

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:

2013/08/21 to 2013/10/16

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

After four weeks, the adult worms were removed from the test vessels. The number of surviving worms (adult mortality) and their biomass change were determined, behavior (including feeding activity) and pathological symptoms were recorded. The adult worms were discarded after counting and weighing.

Vehicle:

no

Details on preparation and application of test substrate:

The composition of the artificial soil was 10 % sphagnum peat; classified according to DIN 11540 (as close to pH 5.5-6.0 as possible, no visible plant remained, finely ground, dried to measured moisture content).

20 % kaolin clay (kaolinite content > 30 %); type: Kaolin W. 0.5 % calcium carbonate. 68.5 % industrial quartz sand; type: Millisil W3, (fine sand is dominant with more than 50 % of the particles between 50 and 200 µm). 1 % food (dried ground horse manure) and deionised water.

The soil was mixed using a KitchenAid mixing machine, with a mixing time: 2 x 2 minutes for each replicate rotations per minute: 240. The amount of soil/test vessel: 675 g wet weight corresponding to 500 g dry weight of artificial soil with a water content corresponding to 40-60 % of WHC. The maximum water holding capacity was WHC (g/100 g soil d.w.): 62.6. The water content at test start: 34.9 - 35.1 % (equivalent to 55.8 – 56.1 % of WHC). At test end: 34.1 – 34.9 % (equivalent to 54.5 – 55.8 % of WHC), with a difference between start and end of the test: max. 1.0 %. The pH at test start was between 6.11 – 6.19, and at test end was 5.68 – 5.80.

One day before test start, the dry artificial soil was pre-moistened by adding deionised water to obtain approximately half of the final water content. Earthworms were acclimatised in a separate batch of the artificial soil (mixed with horse manure) for approximately 24 hours before test start. On the day of the test start, the test item was introduced by dispersing the quantity of test item required to obtain the desired test concentration in the volume of water required to hydrate the soil to 40 60 % of its WHC.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

The test organism was reared under ambient laboratory conditions in the test facility. The breeding medium was a mixture of horse manure, straw, peat (1:1:1), which originated from horse manure and straw were purchased from farmers. The breeding containers were (50 cm x 40 cm x 30 cm), with diffuse light, temperature: about 20 °C and moist soil; pH: about 7. The organisms were adults (approximately 4 months old with clitellum), and they weighed roughly 352 – 498 mg/worm. The time for acclimatization to test conditions was approximately 24 hours in the artificial substrate (with food) , and the food was air-dried and finely ground horse manure.

Study type:

laboratory study

Substrate type:

artificial soil

Remarks:

See preparation of substrate for more details

Limit test:

no

Total exposure duration:

4 wk

Post exposure observation period:

4 and 8 weeks after start of exposure

Test temperature:

18.0 – 21.8°C

pH:

test start: 6.11 – 6.19
test end: 5.68 – 5.80

Moisture:

test start: 34.9 - 35.1 % (equivalent to 55.8 – 56.1 % of WHC)
test end: 34.1 – 34.9 % (equivalent to 54.5 – 55.8 % of WHC)

Details on test conditions:

Light conditions: source: artificial light (Lumilux L58W), with an intensity: 520 lx. The duration of light : dark periods was 16 h : 8 h.

Nominal and measured concentrations:

5.6, 10, 18, 32, 56, 100, 178, 316 mg form./kg soil dry weight

Reference substance (positive control):

yes

Remarks:

The reference item Nutdazim 50 FLOW (Carbendazim, SC 500) was tested in a separate study at concentrations of 5 and 10 mg product/kg soil dry weight.

Key result

Duration:

4 wk

Dose descriptor:

LOEC

Effect conc.:

> 218 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

4 wk

Dose descriptor:

NOEC

Effect conc.:

>= 218 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

8 wk

Dose descriptor:

LOEC

Effect conc.:

69.1 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

8 wk

Dose descriptor:

NOEC

Effect conc.:

38.7 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

8 wk

Dose descriptor:

EC10

Effect conc.:

36.6 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

8 wk

Dose descriptor:

other: EC20

Effect conc.:

53.2 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

Study was performed with the formulated product Propineb WG 70A W.

With the exception of 2.5% mortality at concentrations of 10, 56, 178 and 316 mg form./kg soil dry weight no mortality was observed in any other test item treatment group. 2.5% mortality occurred in the control group. No statistically significant mortality compared to the control was observed at any test item concentration (Fisher’s Exact Binomial Test with Bonferroni Correction, alpha = 0.05, onesided greater). No effects on behaviour (including feeding activity) of the worms were observed during the test.
The test item caused no statistically significant (Williams-t-test, alpha = 0.05, one-sided smaller) change in biomass (change in fresh weight after 4 weeks relative to initial fresh weight) compared to the control group, i.e. a mean weight increase of 30.6% was recorded in the control group and 28.2, 29.0, 31.8, 26.8, 27.6, 26.4, 28.0 and 27.5% at the tested concentrations of 5.6, 10, 18, 32, 56, 100, 178 and 316 mg form./kg soil d.w., respectively. Statistically significant effects (Williams-t-test, alpha = 0.05, one-sided smaller) on number of juveniles compared to the control group were recorded at concentrations of 100, 178 and 316 mg form./kg d.w.

Results with reference substance (positive control):

In a reference test, the number of juveniles was reduced by 39 and 100 % by the toxic standard Nutdazim 50 FLOW (Carbendazim, SC 500) in comparison to the control. Therefore, the observed effects assure a high sensitivity of the test system.

Reported statistics and error estimates:

The endpoints were mortality, change of biomass (difference in fresh weight of surviving worms between test start and four weeks after treatment) and reproduction (the number of juveniles present). The arithmetic mean and the standard deviation per treatment and per control for reproduction, mortality and biomass were calculated. The statistical analysis was performed with the software ToxRat Professional 2.10.06 (Ratte 2010). The EC10 and EC20 (number of juveniles) were estimated by Probit analysis using the maximum likelihood method (Finney 1971). Confidence limits (95 %) of the ECx values were computed by normal approximation. Fisher`s Exact Binomial Test with Bonferroni Correction and the Williams-t-test were used to compare the control with the independent test item groups. For statistical evaluation of the biomass change, the changed mean fresh weight of surviving worms per replicate was used.

Table one

Effects on earthworm reproduction after 56 days

	Propineb  [mg test item /kg dws]
	Control	5.6	10	18	32	56	100	178	316
	Mortality of adult worms after 4 weeks
Mortality [%]	2.5	0	2.5	0	0	2.5	0	2.5	2.5
	Biomass change (change in fresh weight after 4 weeks relative to initial fresh weight)
Mean [%]	30.6	28.2	29.0	31.8	26.8	27.6	26.4	28.0	27.5
	Number of juveniles per surviving adult worm after 8 weeks
Mean	12.9	12.5	12.0	11.9	12.2	11.6	9.0	5.5	2.9
	Number of juveniles per replicate after 8 weeks
Mean ± SD	125.9 ± 23.8	124.8 ± 16.8	117.8 ± 32.6	119.3 ± 18.1	121.8 ± 10.8	113.8  ± 16.5	89.5* ± 16.9	54.0* ± 16.9	28.0* ± 14.5
	Reproduction compared to control [%]
% to control	100	99.1	93.5	94.7	96.7	90.4	71.1	42.9	22.2

 

 

Table two

Effects on mortality, growth and reproduction of the earthworms

Test item Test object Exposure	Propineb  Eisenia fetida Artificial soil
	Adult mortality	Biomass change	Reproduction
	[mg test item /kg dws]
LOEC	>316	>316	100
NOEC	≥316	≥316	56

Validity criteria fulfilled:

yes

Conclusions:

The purpose of this study was to determine the sublethal effects of the formulation on reproduction, mortality and growth of the earthworm Eisenia fetida in a laboratory test. The test was performed according to the recommendations of the OECD Guideline 222 (2004) and the International Standard ISO 11268-2 (1998). Adult earthworms (Eisenia fetida, about 4 months old) were exposed to 5.6, 10, 18, 32, 56, 100, 178 and 316 mg form./kg d.w..
No statistically significantly adverse effects on mortality and biomass of the earthworm Eisenia fetida in artificial soil up to and including 316 mg form./kg soil d.w., i.e. the highest concentration tested, were observed. Statistically significantly adverse effects on reproduction were observed at 100, 178 and 316 mg form./kg soil d.w. Therefore, the No-Observed-Effect- Concentration (NOEC) was determined to be 38.7 mg a.s./kg soil d.w., and the Lowest-Observed- Effect-Concentration (LOEC) was determined to be 69.1 mg a.s./kg soil d.w.

Executive summary:

The purpose of this study was to determine the sublethal effects of the test item on reproduction, mortality and growth of the earthworm Eisenia fetida in a laboratory test. The test was performed according to the recommendations of the OECD Guideline 222 (2004) and the International Standard ISO 11268-2 (1998).

Adult earthworms were exposed to 5.6, 10, 18, 32, 56, 100, 178 and 316 mg form./kg d.w.. Mortality and biomass change were determined after 4 weeks and reproduction was determined after 8 weeks.

The NOEC (reproduction) was determined to be 38.7 mg a.s./kg soil d.w..

Description of key information

The purpose of this study was to determine the sublethal effects of the test item on reproduction, mortality and growth of the earthworm Eisenia fetida in a laboratory test. The test was performed according to the recommendations of the OECD Guideline 222 (2004) and the International Standard ISO 11268-2 (1998). Mortality and biomass change were determined after 4 weeks and reproduction was determined after 8 weeks.

The NOEC (reproduction) was determined to be 38.7 mg a.s./kg soil d.w..

Key value for chemical safety assessment

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

38.7 mg/kg soil dw