REAKTIV-GELB F-68 072 FW

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

10 July 2006 to 30 August 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to OECD test guidance in compliance with GLP and reported with a GLP certificate.

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Deviations:

yes

Remarks:

The pH value was slightly higher than required by the guideline. However, the results of the test have not been impacted.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable.

Analytical monitoring:

not specified

Details on sampling:

Treatment rates: 100 and 1000 mg a.s./kg artificial soil dry weight (dw)

Vehicle:

not specified

Details on preparation and application of test substrate:

The test item was used as a water-miscible substance; therefore, no auxiliary substance was necessary. A stock solution was prepared. All application rates to be tested are based on the content of the active substance (49.0% w/w).

The test item was dissolved in an amount of deionised water sufficient to prepare a stock solution. This stock solution was used to produce one dosage solution of the test item. An appropriate amount of the stock and the dosage solution respectively served to prepare the different application rates of the test item onto the artificial soil.

The test item was incorporated into the soil by thoroughly mixing in the respective test item solution for approx. 10 minutes.

The method of pre-treatment of the test item was selected according to the characteristics of the test item and is described in the test guideline.

The worms were introduced into the test vessels, after the treated artificial soil was filled into the test vessels. The test animals were exposed to the test Item by dermal and alimentary uptake.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

The earthworms used in this test, Eisenia fetida (Lumbricidae, Earthworms) are kept at ECT Oekotoxikologie GmbH since February 1994. They were originally delivered by Co. Landenberger (D-72355 Sehflmberg).

Only adult worms (with clitellum) with a fresh weight between 300 and 600 mg were used. The selected test animals were acclimatised in untreated artificial soil at least 24 hours prior to the start of the test.

Breeding and keeping of the test organisms is described in detail in the Standard Operation Procedures (SOP) A 7.1 operated by the laboratory except those points which are otherwise outlined in this report.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

yes

Total exposure duration:

14 d

Post exposure observation period:

No post exposure observation period

Test temperature:

19.8 - 21.1 °C (recommended 20 ± 2 °C)

pH:

6.7-6.8 (recommended 6.0 ± 0.5)

Moisture:

51.7 -57.7% (recommended 40 - 60% of the WHCmax)

Details on test conditions:

Test Units
Test vessels were 1.5 L preservation Jars ("Tulpenform"), Co. Week, Wehr-Oflingen. The test vessels were covered by glass lids.

Test Conditions
The earthworms were tested in an artificial soil. The composition of this artificial soil was based on OECD Guideline No. 207 (1984) (the percentages refer to the dry weight);

10% Sphagnum peat (no visible plant remains; finely ground and air-dried)
20% Kaolln-Clay (kaolinite content > 30%)
69.5% Quartz sand (fine sand content with particles between 50 and 200 μm higher than 50%)
ca, 0.5% Calcium carbonate (CaCO3)

The air dried peat was shredded in a chaff-cutter. Afterwards, all parts of the artificial soil were mixed homogeneously. 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 artificial soil was determined by drying a small sample at 105 °C.

The following conditions were observed:
Temperature: 20 ± 2 °C
Light cycle: continuous light
Feeding: none

Chronological description of the test performance including methods used:

Performance of the test with two concentrations and an untreated deionised water control. Per concentration and control four test vessels (= replicates) each containing 10 earthworms were used.

Preparation of the artificial soil:
The dry constituents of the artificial soil were prepared. Calcium carbonate was used to adjust the pH-value to 6 ± 0.5. The maximum water capacity (WHCmax) of the artificial soil was determined. One day before starting the test the artificial soil (per replicate 500 g dw) was pre-moistened with deionised water (40 - 60% of WHCmax minus volume of application solution).

Selection of worms:
Adult worms were selected out of the culture in order to acclimatise them for at least 24 h in untreated artificial soil.

Test preparation;
Preparation of the test item solutions. The test item was dissolved in a sufficient amount of deionised water to prepare a stock solution. This stock solution was diluted with deionised water to obtain one test item solution (= dosage solution) for each test item concentration.
Filling in of the artificial soli into the test containers (corresponding to 500 g dw). In each of the test containers 100 mL of the aqueous test item solutions was applied onto the pre-moistened artificial soil. This amount of water/test item-mixture served also to adjust the final soil moisture of 40- 60% of WHCmax, In the controls 100 mL deionised water was added.
Mixing of the artificial soil until the test Item solution was homogeneously distributed. Determination of the pH-value and the moisture of the artificial soil once in each concentration (samples were taken from one test vessel).

Introduction of worms Into the test containers:
The acclimatised worms were weighed (individually) immediately after application of the test item. Sets of 10 worms were placed onto the soil surface of each test container randomly.
The test vessels were kept Into an incubator or a climate chamber with controlled abiotic conditions (esp. temperature).

Day 7:
The worms were removed carefully from their test containers. During this process morphological and behavioural changes and the number of surviving worms in each test container were recorded. Worms were classified as dead when they did not respond to a gentle mechanical stimulus to the front end. The combined weight of surviving worms from each replicate was determined. Afterwards the worms were put back onto the soil surface in the containers.

Day 14:
The worms were removed carefully from the test containers. During this process morphological and behavioural changes and the number of surviving worms in each test container were recorded. Worms were classified as dead when they did not respond to a gentle mechanical stimulus to the front end. The individual weight of the surviving worms from each replicate was determined.
Determination of the pH-value and the moisture of the artificial soil once in each concentration (samples were taken from one test vessel).

Nominal and measured concentrations:

Treatment rates: 100 and 1000 mg a.s./kg artificial soil dry weight (dw)

Reference substance (positive control):

yes

Remarks:

chloroacetamide

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Conc. based on:

act. ingr.

Basis for effect:

growth

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

0.0% mortality was observed at the control and at the concentration of 1000 mg a.s./kg artificial soil (dw). 2.5% mortality was observed at the concentration of 100 mg a.s./kg artificial soil (dw).

Other effects on the morphology and the behaviour of the worms have not been observed.

Results with reference substance (positive control):

Reference Test
Dates of work: April 04, - April 18,2006.
Mortality: In the control, 0% of the adult worms died. At the concentrations of 11.2 and 19.3 mg/kg soil (dw) 2.5% mortality was observed. At the concentration of 33.5 mg/kg soil (dw) 60% mortality was observed.
Comments: The LCgo value for the toxic reference item, chloroacetamide, was determined as 31.7 mg/kg. This value is slightly above the historical range of the results of the ECT laboratory (mean LC50 value = 21.7 ± 5.8 mg/kg) but within the upper (36.6 mg/kg ) and lower (12.0 mg/kg) warning limit. The warning limits were calculated as two standard deviations from the historical mean of tine LCso (all calculated using the log concentration: according to Environment Canada 1999). In addition, the LCh value is within the order of magnitude published in scientific literature (EC 1984, Kula 1998).

Reported statistics and error estimates:

Biomass data were checked for normal distribution by R/s test procedure and homogeneity by Cochran's test. Treatment means were compared by ANOVA and Student t-test and tested for statistically significant (p ≤ 0.05) differences compared to the control (Sachs 1982).

The statistical software package ToxRat Professional 2.09 was used for these calculations.


Statistical analysis (Student-t test; 2-sided, p s 0.05) showed no significant difference in the biomass development of Individual adults after 14 days between the control and the two concentrations of the test item tested.

Number and biomass (mg) of the earthworms determined at the beginning, after 7 and 14 days of the test. The concentration is given in mg a.s./kg (SD = Standard Deviation; I = Individual Weight).

Day	Parameter	Control				100				1000
		a	b	c	d	a	b	c	d	a	b	c	d
0	Weight (I)	442	377	394	330	507	473	450	476	423	45	394	451
		431	367	387	336	428	447	366	449	486	447	421	387
		463	428	354	354	419	481	340	388	477	375	376	487
		382	504	495	360	366	367	341	345	444	428	426	342
		517	491	370	491	393	417	358	512	525	406	375	389
		437	502	380	351	446	355	360	373	393	361	387	347
		446	422	398	433	465	437	496	356	492	411	390	363
		394	356	355	417	497	434	338	355	443	408	388	410
		425	378	446	352	508	416	413	350	375	432	458	412
		427	352	404	356	377	350	518	352	352	344	378	378
	Number Average SD	10	10	10	10	10	10	10	10	10	10	10	10
		436.4	417.7	398.3	378.0	439.6	417.7	398.0	395.6	441.0	407.7	399.3	396.6
		37.0	61.4	43.2	51.8	52.6	46.7	67.7	60.7	56.7	38.2	27.1	45.5
7	Weight (sum)	4564	4499	4376	4366	4795	3745	4420	4164	4002	3677	4047	4233
	Number Average	10	10	10	10	10	10	10	10	10	10	10	10
		456.4	449.9	437.6	436.6	479.5	416.1	442.0	416.4	400.2	367.7	404.7	423.3
14	Weight (I)	487	405	352	473	410	444	375	381	418	330	377	371
		468	372	378	405	466	464	400	494	523	412	424	404
		455	369	377	346	415	411	394	367	469	391	375	400
		380	450	356	379	400	437	409	365	340	360	402	388
		443	331	378	376	335	450	418	352	310	367	390	375
		405	419	400	482	447	500	420	540	318	354	352	372
		395	475	382	325	412	363	393	465	415	369	446	488
		438	469	388	380	519	442	466	319	408	365	360	378
		378	426	449	411	513	411	454	372	410	344	379	402
		384	417	424	421	495	-	620	374	407	310	368	516
	Number Average SD	10	10	10	10	10	9	10	10	10	10	10	10
		423.3	413.3	388.2	399.8	441.2	435.8	434.9	402.9	401.8	360.2	387.3	407.4
		39.8	45.9	29.6	50.1	58.0	38.3	70.7	71.2	65.9	28.9	29.3	47.6

Worm dead

Observations: None

Validity criteria fulfilled:

yes

Conclusions:

The NOEC was regarded as ≥ 1000 mg a.s./kg artificial soil (dw).

Executive summary:

Study conducted to OECD Guideline No. 207 for the Testing of Chemicals "Earthworm, Acute Toxicity Tests" adopted April 4,1984. Deviations: The pH value was slightly higher than required by the guideline. However, the results of the test have not been impacted. Study in compliance with the Principles of Good Laboratory Practice (GLP) and reported with a GLP certificate.

 

Findings: 0.0% mortality was observed at the control and at the concentration of 1000 mg a.s./kg artificial soil (dw). 2.5% mortality was observed at the concentration of 100 mg a.s./kg artificial soil (dw).

 

Other effects on the morphology and the behaviour of the worms have not been observed.

 

The NOEC was regarded as ≥1000 mg a.s./kg artificial soil (dw).

 

Description of key information

The NOEC was regarded as ≥ 1000 mg a.s./kg artificial soil (dw).

Key value for chemical safety assessment

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

1 000 mg/kg soil dw

Additional information

A study has been conducted according to OECD Guideline No. 207 for the Testing of Chemicals "Earthworm, Acute Toxicity Tests" adopted April 4,1984. Deviations: The pH value was slightly higher than required by the guideline. However, the results of the test have not been impacted. Study in compliance with the Principles of Good Laboratory Practice (GLP) and reported with a GLP certificate.

 

Findings: 0.0% mortality was observed at the control and at the concentration of 1000 mg a.s./kg artificial soil (dw). 2.5% mortality was observed at the concentration of 100 mg a.s./kg artificial soil (dw).

 

Other effects on the morphology and the behaviour of the worms have not been observed.