Chlorothalonil

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:

18 Apr 2001 to 15 Aug 2001

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Version / remarks:

1984

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

yes

Remarks:

acetone

Details on preparation and application of test substrate:

An amount of the test item sufficient to prepare a stock solution was dissolved in acetone. Aliquots of this stock solution were further diluted with acetone to produce the other required dosage solutions of the test item. An appropriate amount of the stock or other dosage solution was used to soak a portion of the quartz sand of the artificial soil. After evaporation of the solvent by placing under a fume hood, the quartz sand was thus coated by the test item. The test item coated fraction of the quartz sand was than incorporated into the remainder of the artificial soil by thoroughly mixing it in for approximately 10 minutes, as described in the guideline (ISO 1998). This procedure was done for each test vessel individually.

Each mixture was stirred for 10 minutes. 12.5 mL of each mixture was applied onto 50 g quartz sand for each replicate vessel individually; for the solvent control 12.5 mL acetone was applied onto 50 g quartz sand. After evaporation, each 50 g of treated sand was mixed into the prepared test soil to give a total dry weight equivalent of 500 g soil for the replicate.

Test organisms (species):

Eisenia andrei

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earthworms
- Age: The worms were at least two months, but not older than one year old, however the age of individuals did not differ by more than four weeks. Animals fulfilling these requirements were bred in a special synchronisation culture.
- Selection criteria: Only adult worms (with clitellum) with a fresh weight (FW) between 300 and 600 mg were used.

CULTURING
The earthworms were kept at supplier's 1987 until Feb. 1994. Since February, 1994 the worms are kept at at the test facility.
- Artificial soil properties: The earthworms were bred in a mixture of artificial soil and food (1 - 30 vol. %). Details on the artificial soil are given in 'Details on test conditions'.
- Temperature: 15 - 30 °C
- Light cycle: Permanent dark
- Feeding: According to demand; usually once per week.

ACCLIMATION
The worms selected for the test were acclimatised in artificial soil under test conditions for at least 24 hours before starting the test.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Test temperature:

20- 22 °C

pH:

- pH at test initiation: 6.0
- pH at test termination: 5.3 - 5.4

Moisture:

44.8 - 51.4 % of the dry weight

Details on test conditions:

TEST SYSTEM
- Test container: Bellaplast container: Area 11 cm x 15.5 cm, height 6 cm, transparent cover
- Amount of soil or substrate: 500 g
- No. of organisms per container: 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 4
- No. of replicates per vehicle control: 4
- Feeding: finely ground com meal, stinging nettle and bran powder

ARTIFICIAL SOIL PROPERTIES
The artificial soil was composed as follows (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%)
- 68 - 69% Quartz sand (fine sand content with particles between 50 and 200 microns higher than 50%)
- ca. 1% Calcium carbonate (CaC03)
The air dried peat was shredded in a chaff-cutter. Afterwards, all parts of the artificial soil were mixed until homogeneous. The moisture was adjusted to 50 ± 10% of dry weight using deionised water. The pH value was adjusted to 6 ± 0.5 using calcium carbonate. Food of a good quality shown to be suitable for at least maintaining worm weight during the test was used (e g. finely ground com meal, stinging nettle and bran powder). The amount of food in the breeding substrate was approximately 5 %.

OTHER TEST CONDITIONS
- Photoperiod: 16:8 h
- Light intensity: 400- 800 lx

EFFECT PARAMETERS MEASURED: mortality, biomass development, behavioural changes, reproduction
- Mortality: Adult mortality was assessed at 28 days after treatment. Worms were classified as dead when they did not respond to a gentle mechanical stimulus to the front end.
- Biomass development: The adult worms were weighed individually as follows. The artificial soil of an acclimatisation container or of each test container was put on a steel tray. The worms were carefully removed from the soil and transferred into a dish with reconstituted water to remove soil particles. After a short period of time (less than 10 min) they were laid on a piece of tissue paper to remove the water drops. Thereafter the worms were weighed individually (day 0 and day 28).
- Behaviour and morphology changes: Changes in the behaviour (e g. food consumed) and the morphology of the adult worms were recorded at 28 days. Additionally, the presence of cocoons was determined by classifying their occurrence in a "yes/no" manner. Both examinations were done when the artificial soil of a test vessel was thinly spread out on a steel tray.
- Number of juveniles: Number of juvenile earthworms: The juvenile worms were extracted from the soil after 56 days by keeping the test container in a warm water bath (50 - 60 °C) for at least 20 minutes. The worms were collected from the soil surface and counted. After the extraction, the substrate of one randomly collected test container per concentration was examined for remaining juveniles to assess the extraction efficiency.

VEHICLE CONTROL PERFORMED: yes

Nominal and measured concentrations:

- Nominal test concentrations: Blank control, solvent control, 2, 5, 10, 20 and 50 mg a.i./kg.

Reference substance (positive control):

yes

Remarks:

Benomyl was tested in a separate study

Key result

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

>= 50 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- Mortality: Only 2.5% mortality was observed in the control and no mortality with the solvent control and all concentrations of the test item tested. Therefore, no statistical analysis was carried out and the LC50 value could not be calculated.
- Adult biomass: The Cochran's test confirmed the homogeneity of the data. The ANOVA and the DunnettTest showed no significant difference (p 0.05) in the biomass of the adult worms between the control and all concentrations of the test item tested at day 28. The mean percentage of initial weight at Day 28 were determined to be 90.1, 88.8, 88.0, 94.5, 90.7, 89.9 and 88.3% for the blank control, solvent control, 2, 5, 10, 20 and 50 mg a.i./kg treatments, respectively The results are tabulated in Table 1 in 'Any other information on materials and methods incl. tables.'
- Reproduction: The Cochran’s test confirmed the homogeneity of the data. The ANOVA and the Dunnett’s test (2-sided, p 0.05) showed no significant difference in the number of juveniles between the control and all concentrations of the test item tested. Therefore, the NOEC was considered to be 50 mg a.i./kg (the highest concentration tested). The results are tabulated in Table 2 in 'Any other information on materials and methods incl. tables.'
- Food consumption: Most of the food was consumed by the worms in all treatments. In those cases where food was not consumed totally when checking the feeding rate, the newly given amount was reduced on demand. No correlation between a decrease in food consumption and chemical concentration was observed. No other effects on behaviour or morphology of the adult earthworms were observed.

Results with reference substance (positive control):

In the test with the reference item benomyl, a significant reduction was found at the concentrations of 3 and 5 mg a.i./kg. Therefore, the NOEC was derived to be 1.0 mg a.i./kg. The observed effect is within the expected range from literature (ISO 1998). The effects of benomyl confirm suitable sensitivity of the test system.

Reported statistics and error estimates:

The U-test according to Mann-Whitney was used to compare the solvent control with the water control with respect to the biomass and the reproduction. Cochran's-test was used to confirm homogeneity (p 0.05). A One-Way Analysis of Variance (ANOVA), followed by a Dunnett’s test (2-sided, p 0.05) was used to determine whether or not there were significant differences in the mean loss/increase in biomass of the adult worms (measured as change between the weight at day 0 and day 28 for each concentration) between each concentration and the control. The same method was used to calculate whether there were significant differences between the number of juveniles in the controls and in the treated vessels at day 56. Based on the results of these ANOVA calculations and taking into consideration the behavioural and morphological effects, the NOEC (= No Observed Effect Concentration) was determined. The statistical software package SPSS 7.5 was used for these calculations.

Table 1: The biomass (mg) of the adult earthworms determined at the beginning and after 28 days of the test. All concentrations are given mg a.i./kg.

 

Day	Parameter	Control				Solvent Control				2 mg a.i./kg				5 mg a.i./kg				10 mg a.i./kg				20 mg a.i./kg				50 mg a.i./kg
1	Weight	458	385	300	471	512	479	414	544	449	422	491	381	582	321	385	432	462	499	544	425	358	389	310	410	441	412	459	300
		561	475	385	463	414	570	448	355	367	488	403	441	496	400	381	347	544	400	436	367	476	580	563	414	504	405	444	413
		504	516	565	382	444	511	396	397	461	412	429	451	447	337	410	384	443	449	387	354	457	582	501	300	441	536	428	414
		366	378	380	378	489	370	300	433	430	582	354	315	414	565	325	318	390	307	346	489	461	400	483	424	326	445	340	379
		554	390	425	320	434	461	511	389	422	360	365	444	364	464	577	356	491	549	382	375	538	508	417	370	368	424	382	342
		441	488	403	502	540	394	310	450	547	390	552	395	417	357	354	316	362	479	418	345	440	493	440	480	554	402	501	396
		423	467	529	388	406	402	518	323	302	388	446	388	381	465	383	300	397	320	522	393	418	339	334	468	532	536	589	300
		362	523	503	323	514	472	588	584	480	442	300	497	477	399	536	412	442	557	437	411	556	337	365	404	494	377	354	529
		500	409	430	413	556	357	442	382	418	500	473	446	461	600	530	405	412	572	357	429	418	355	390	476	329	406	385	383
		340	356	442	574	413	418	364	391	600	460	473	405	455	545	402	386	593	333	495	442	445	426	452	380	493	481	504	342
	Number	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
	Average	450.9	438.7	436.2	421.4	472.2	443.4	429.1	424.8	447.6	444.4	428.6	416.3	449.4	445.3	428.3	365.6	453.6	446.5	432.4	403	456.7	440.9	425.5	412.6	448.2	442.4	438.6	379.8
	SD	79.1	61.7	78.4	80.7	56.5	67.2	92.4	82.0	84.2	65.8	74.7	50.4	62.3	98.9	86.6	45.1	72.0	101.5	68.6	44.8	57.7	94.1	78.5	55.2	82.4	56.7	77.7	67.0
28	Weight	508	354	393	408	329	372	465	377	275	409	459	468	308	390	433	307	342	396	361	454	429	426	373	495	329	407	425	334
		363	562	311	376	271	423	384	384	450	492	442	468	363	504	504	339	380	430	453	379	376	257	323	413	405	291	482	337
		402	351	476	348	456	381	394	491	551	404	383	437	344	358	330	396	375	402	401	429	472	537	451	321	307	502	517	316
		391	393	323	386	557	395	234	425	497	368	316	361	466	390	453	384	440	387	418	344	470	324	427	491	427	386	443	272
		428	338	445	484	437	357	366	411	541	271	404	324	392	490	350	382	512	464	334	333	414	378	423	385	476	450	319	246
		472	441	379	422	469	443	320	304	304	341	306	372	455	487	381	379	313	371	429	566	330	355	412	300	380	403	344	291
		371	386	425	358	423	302	525	405	238	319	374	293	302	334	433	486	333	401	388	388	347	298	404	403	450	365	371	291
		445	304	344	316	428	404	355	373	309	298	370	281	534	465	408	386	458	399	375	286	361	438	374	361	361	381	355	350
		400	374	367	400	534	367	327	410	378	374	324	324	425	363	478	272	518	356	385	406	346	344	333	430	278	392	407	571
		421	385		309	360	362	501	200	369	513	355	519	305	415	433	325	459	222	324	327	362	410	441	363	355	383	296	401
	Number	10	10	9	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
	Average	420.1	388.8	384.8	380.7	426.4	380.6	387.1	378	391.2	378.9	373.3	384.7	389.4	419.6	420.3	365.6	413	382.8	386.8	391.2	390.7	376.7	396.1	396.2	376.8	396	395.9	340.9
	SD	45.2	71.0	55.8	52.1	87.9	39.1	88.9	78.2	112.9	78.6	51.3	82.9	79.2	62.2	54.4	58.9	74.3	63.8	40.7	79.7	52.1	80.2	43.9	64.6	63.4	54.6	71.6	91.9

 

Table 2: Number of offspring after 56 days

Treatment	Replicate	Number of Juveniles	Number of Juveniles per Treatment (mean ± sd)	Percentage of Control
Control	A	28	45.5 ± 12.2	100
Control	B	50
Control	C	48
Control	D	56
Solvent Control	A	63	46.3 ± 14.9	101.6
Solvent Control	B	53
Solvent Control	C	29
Solvent Control	D	40
2 mg a.i./kg	A	51	55.5 ± 17.4	122
2 mg a.i./kg	B	66
2 mg a.i./kg	C	72
2 mg a.i./kg	D	33
5 mg a.i./kg	A	45	35.3 ± 9.6	77.5
5 mg a.i./kg	B	37
5 mg a.i./kg	C	22
5 mg a.i./kg	D	37
10 mg a.i./kg	A	53	56.0 ± 10.8	123.1
10 mg a.i./kg	B	49
10 mg a.i./kg	C	72
10 mg a.i./kg	D	50
20 mg a.i./kg	A	45	47.5 ± 20.0	104.4
20 mg a.i./kg	B	75
20 mg a.i./kg	C	27
20 mg a.i./kg	D	43
50 mg a.i./kg	A	32	34.0 ± 10.4	74.7
50 mg a.i./kg	B	25
50 mg a.i./kg	C	30
50 mg a.i./kg	D	49

Validity criteria fulfilled:

yes

Remarks:

See 'Any other information on materials and methods incl. tables'

Conclusions:

The 56-d NOEC was determined to be 50 mg/kg soil dw (highest concentration tested).

Executive summary:

Reproduction and chronic toxicity of the test substance (purity 98.7%) was tested on the earthworm Eisenia andrei according to the ISO 11268-2 Part 2 (1998) and in compliance with GLP. Worms of unknown age but with clitellum (between 2 months and one year) and of the same age group were obtained from a laboratory culture. Weights of the worms varied between 300 and 600 mg. Worms were acclimatized for at least 24 hours before start of the test. The five test concentrations were 2, 5, 10, 20 and 50 mg/kg dw of artificial soil (10% peat, 20% kaolin clay, 68 - 69% quartz sand and ca. 1% calcium carbonate). Moisture of the artificial soil was readjusted to 50 ± 10% of dry weight. Acetone was used as a solvent. Soil was moistened at weekly term. Tests were performed under a 16:8 light/dark cycle (400-800 Lux). There were four replicates per concentration, each replicate contained 10 worms. A water treated control was included in the tests. Benomyl was used as the toxic reference. Test parameters were mortality (assessed at 28 days after application), biomass development (day 0 and 28), behaviour or morphology and reproduction (after 56 days).

 

Adult mortality was 2.5% in the control and no mortality was observed at any of the tested concentrations and in the solvent control. With ANOVA and the Dunnett’s-Test, no significant differences in the biomass nor reproduction were observed between the control and the five concentrations of the test substance. The NOEC for biomass and reproduction was determined by the author at 50 mg/kg. There were no effects on morphology, food consumption and behaviour. The effects of Benomyl confirmed the sensitivity of the test system.

Description of key information

56-d NOEC = 50 mg/kg soil dw based on reproduction, Eisenia andrei, ISO 11268-2 (Part 2), Moser 2001

Key value for chemical safety assessment

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

50 mg/kg soil dw

Additional information

Reproduction and chronic toxicity of the test substance (purity 98.7%) was tested on the earthworm Eisenia andrei according to the ISO 11268-2 Part 2 (1998). Worms of unknown age but with clitellum (between 2 months and one year) and of the same age group were obtained from a laboratory culture. Weights of the worms varied between 300 and 600 mg. Worms were acclimatized for at least 24 hours before start of the test. The five test concentrations were 2, 5, 10, 20 and 50 mg/kg dw of artificial soil (10% peat, 20% kaolin clay, 68 - 69% quartz sand and ca. 1% calcium carbonate). Moisture of the artificial soil was readjusted to 50 ± 10% of dry weight. Acetone was used as a solvent. Soil was moistened at weekly term. Tests were performed under a 16:8 light/dark cycle (400 - 800 Lux). There were four replicates per concentration, each replicate contained 10 worms. A water treated control was included in the tests. Benomyl was used as the toxic reference. Test parameters were mortality (assessed at 28 days after application), biomass development (day 0 and 28), behaviour or morphology and reproduction (after 56 days).

 

Adult mortality was 2.5% in the control and no mortality was observed at any of the tested concentrations and in the solvent control. With ANOVA and the Dunnett’s-Test, no significant differences in the biomass nor reproduction were observed between the control and the five concentrations of the test substance. The NOEC for biomass and reproduction was determined by the author at 50 mg/kg. There were no effects on morphology, food consumption and behaviour. The effects of Benomyl confirmed the sensitivity of the test system.