Diquat dibromide

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:

04 Nov 2005 to 22 Nov 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

- Preparation of the test substrate: The dry constituents are blended in the correct proportions (10% of peat, 20% of kaolin and 70% of sand). Bideionized water is added to give an overall moisture content of about 40% of the WHOmax. The complete mixture should be moist but not so wet that water appears when the artificial soil is compressed. The initial pH of the soil were 6.67 and the initial and final humity of the soil was 0.095 g water/g soil and 0.062 g water/g soil, respectively.

- Preparation of the test concentration: The test concentrations were prepared by add the test substance directly to the soil. Each test concentration was prepared by adding a mass of the test substance to 750 g of artificial soil. The flask used to weight the test substance was rinsed 3 times with a total of 5 mL of bideionized water and it was incorporated to the respective soil.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earch worm
- Source: commercial culture
- Culture condition: Earthworms are grown in dried manure substrate and fed with fruits and vegetables
- Age at test initiation: > 2 months (sexually developed)
- Feeding during test: without food

ACCLIMATION
- Acclimation period: > 2 weeks

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

14 d

Test temperature:

18.1 - 20.7 °C

pH:

6.67

Moisture:

Approximately 40% of the WHOmax
- Initial of the test: 0.095 g water/g soil
- End of the test: 0.062 g water/g soil

Details on test conditions:

TEST SYSTEM
- Test vessel: 1.2 L glass flasks
- Total weight: Approximately 750 g
- No. of organisms per container: 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 4

SOURCE AND PROPERTIES OF SUBSTRATE: See 'Details on preparation and application of test substrate'

OTHER TEST CONDITIONS
- Photoperiod: 24 h of attenuated light

EFFECT PARAMETERS MEASURED
Test organisms were observed after 14 days of exposure and a count was conducted on the number of dead and alive organisms. Definitive test observations are summarized in Final results based on those observations was applied the statistic procedure of Trimmed Spearman Karber.

RANGE FINDING STUDY
In order to determine the best working range a 96-h preliminary test was conducted with four concentrations plus one control and five organisms per concentration with one replication.
- Test concentration: 1, 10, 100 and 1000 µL of test substance/kg soil
- Conclusion: Results show that the 14 days effects should start at concentrations > 1000 mg/kg of soil

Nominal and measured concentrations:

- Nominal concentration: 35.17, 73.90, 149.28, 295.98 and 587.19 mg a.i. (as cation)/kg soil (See effective concentration analysis and Table 1 in 'Any other information on materials and methods incl. tables')

Reference substance (positive control):

no

Key result

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

176.21 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

94.33 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

cation

Basis for effect:

mortality

Remarks on result:

other: Original value presented in study

Remarks:

95% C.L.: 77.56 - 114.73 mg a.i./kg dry soil

Details on results:

After 14 days exposure to the test substance, all organisms survived in the control and 35.17 mg a.i./kg soil treatment group. The concentration that immobilizes 100% of organisms was not determinated in this study. The average mortality were 50%, 65%, 90% and 90% in 73.9, 149.28, 295.98 and 587.19 mg a.i.(as cation)/kg soil dw, respectively. Based on the findings, the LC50 was determined to be 94.33 mg cation/L (equivalent to 176.21 mg registered substance/L).

Reported statistics and error estimates:

The results were treated according to the Trimmed-Spearman-Karber method for determination of the LD50 and of the reliability intervals to 95% when observed mortality for over 50% of the organisms under exposure in the highest test concentration. The reliability interval that was not achieved with this method was estimated according to Hoeven.

Table 2. Test concentration and observed effects in 14 days exposition

Identification	Test concentration (mg a.i. (as cation) /kg soil)	Observed effects - 14 days (Dead/live)
		Replication 1	Replication 2	Replication 3	Replication 4
A	-	0/10	0/10	0/10	0/10
B	35.17	0/10	0/10	0/10	0/10
C	73.9	8/2	8/2	0/10	4/6
D	149.28	6/4	9/1	5/5	6/4
E	295.98	9/1	9/1	8/2	10/0
F	587.19	10/0	9/1	9/1	8/2

Calculation of key result

The original effect levels were expressed as cation species of the registered substance. The key effect levels are re-calculated and corrected to include the counterion species by multiplying with 1.868 (344.0 g/mol molecular weight of registered substance divided by 184.2 g/mol molecular weight of cation species).:

1.868 x 94.33 mg cation/kg soil dw = 176.21 mg registered substance/kg soil dw

Validity criteria fulfilled:

yes

Conclusions:

Based on the findings, the LC50 was determined to be 94.33 mg cation/L.

Executive summary:

A laboratory study was conducted to determine the acute toxicity of the test substance to soil earthworm (Eisenia foetida). The study was based on OECD TG 207 and in compliance with GLP criteria. The study was performed with five test concentrations (35.17, 73.90, 149.28, 295.98 and 587.19 mg a.i. (as cation)/kg soil) and a control group. Each concentration has four replications with ten organisms per replicate. Thus, in total of 40 organisms per concentration. The test substrate was an artificial soil prepared with 70% sand, 20% kaolin and 10% peat. The test was conduced under continuous and attenuate illumination at a temperature of 20 ± 2°C. The end point was based on mortalities after 14 days. The concentrations of active ingredient (expressed as cation) were determined analytically.

After 14 days exposure to the test substance, all organisms survived in the control and 35.17 mg a.i./kg soil treatment group. The concentration that immobilizes 100% of organisms was not determinated in this study.The average mortality were 50%, 65%, 90% and 90% in 73.9, 149.28, 295.98 and 587.19 mg a.i.(as cation)/kg soil dw, respectively. Based on the findings, the LC50 was determined to be 94.33 mg cation /kg of soil dw.

Reference 2

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Jun 2007 to 20 Jul 2007

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:

2004

Deviations:

not specified

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

- Preparation of the test substrate: Approximately 24 hours before test start, the artificial soil was prepared for use. Deionised water was added to the dry components to adjust the water content to approximately 40 - 60 % of WHC. Adult earthworms of the species Eisenia fetida (approximately 3 months old with clitellum) were acclimatised in a separate batch of the artificial substrate, mixed with horse manure for approximately 24 hours before test start.
- Application of the test substrate: The test substrate consisted of artificial soil, the test item (except in controls) and deionised water. The test item was introduced by dispersing the quantity of the test item to obtain the desired test concentration in a portion of deionised water necessary to wet the soil substrate to the required water content. The control substrate contained the corresponding amount of deionised water only. Each test vessel was then filled with the treated soil (approximately 751 g wet weight corresponding to 556 g dry weight). After a randomising procedure according to the worm fresh weight, selected groups of 10 worms were then randomly assigned to each treatment group. The individually weighed worms (10 worms/vessel) were placed on the surface of the soil. The test vessels were closed with perforated transparent lids, which allowed for gas exchange between substrate and atmosphere and access of light to prevent worms from escaping. The test vessels were then set up at random in a controlledenvironment test room.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earthworm
- Source: Reared under ambient laboratory conditions in the test facility
- Breeding medium: Mixture of horse manure, straw, peat (1:1:1)
- Breeding conditions: 50 cm x 40 cm x 30 cm breeding containers, with moist soil, under diffuse light, approximately 20 °C and pH 7 condition.
- Age at test initiation: Adult worms (approximately 3 months old with clitellum)
- Weight of animals used in the test: 304 - 463 mg/worm
- Food used during the test: Air-dried and finely ground horse manure
- Feeding frequency: Weekly during the first four weeks of the test
To avoid systematic errors in distributing the worms to the test vessels the homogeneity of the test population was being determined by individually weighing of worms sampled randomly from the synchronized population, having ensured homogeneity, batches of worms were then selected, weighed and assigned to test vessels using a randomisation procedure.

ACCLIMATION
- Acclimation period: Approximately 24 hours in the artificial substrate

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

8 wk

Remarks:

4 weeks adult mortality; 4 weeks juvenile development

Test temperature:

18 - 21 °C

pH:

- Test start: 6.2 - 6.3
- Test end: 5.9 - 6.0

Moisture:

- Test start: 34.8 - 35.2% (equivalent to 56.9- 57.5% of WHC)
- Test end: 34.4 - 34.9% (equivalent to 56.2 - 57.0% of WHC)

Details on test conditions:

TEST SYSTEM
- Test container: Plastic vessel (18.3 cm x 13.6 cm x 6.4 cm) with a lid which is pervious to air and light
- Amount of soil or substrate: Approximately 751 g wet weight
- No. of organisms per container: 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8

SOURCE AND PROPERTIES OF SUBSTRATE
- Details of the subtrate preparation see 'Details on preparation and application of test substrate'
- Peat: 10% sphagnum peat
- Clay: 20% kaolinite clay
- Calcium carbonate: 0.4% calcium carbonate
- Sand: 69.6 % industrial quartz sand
- WHCmax: 61.2 %

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness
- Light intensity: 590 lux

EFFECT PARAMETERS MEASURED
Behaviour (worms on the soil surface) and feeding activity were assessed during each feeding in weekly intervals. During assessment of the feeding activity the percentage of manure consumed per replicate was determined visually. The water content of the soil was checked weekly by reweighing of the test vessels. Water loss was compensated, if necessary. 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, behaviour (including feeding activity) and pathological symptoms were recorded. The adult worms were discarded after counting and weighing. Subsequently, the soil of each vessel was mixed carefully with 5 g manure. This was the last feeding occasion during experiment. The test was then continued for another four weeks. The final assessment included counting of surviving juveniles per test vessel, determination of the water content and pH measurements of the artificial soil. Juveniles were counted by manual inspection of the substrate.

The following parameters were studied during the test:
At test started :
- individual fresh weight (mg/worm)
- behaviour of earthworms
- determination of physico-chemical parameters of the artificial soil (water content, pH)

Weekly:
- observation of behavioural and pathological symptoms (including feeding activity)

4 weeks after test start:
- number of surviving adult earthworms per replicate
- observation of behavioural and pathological symptoms
- fresh weight of surviving adult earthworms per replicate

8 weeks after test start:
- number of surviving juveniles per replicate
- observation of behavioural and pathological symptoms
- morphological alterations
- determination of physico-chemical parameters (water content, pH)

Nominal and measured concentrations:

- Nominal concentration: 0 (negative control), 5, 10, 17, 27, 37 mg cation/kg soil dw.

Reference substance (positive control):

no

Key result

Duration:

8 wk

Dose descriptor:

NOEC

Effect conc.:

69.1 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Mortality, behaviour, biomass and reproduction

Remarks on result:

other: Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation

Duration:

8 wk

Dose descriptor:

NOEC

Effect conc.:

37 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

cation

Basis for effect:

other: Mortality, behaviour, biomass and reproduction

Remarks on result:

other: Original value presented in study

Details on results:

An overview of the results is provided in Table 1 and Table 2 in ‘Any other information on results incl. tables’.

No mortality (0 %) occurred in the control group. The test item caused no mortality at any tested concentration after 4 weeks. No effects on behaviour (including feeding activity) of the worms were observed during the test. The test item caused no statistically significant change in biomass (change in fresh weight after 4 weeks relative to initial fresh weight) compared to the control group at all the concentrations tested, i.e. a weight increase of 17.9, 19.6, 16.5, 19.0, 17.2 and 15.3 % was recorded in the control group and at concentrations of 5, 10, 17, 27 and 37 mg cation/kg soil d.w., respectively. No statistically significant effects (DUNNETT-Test, p > 0.05) on the number of juveniles compared to the control group were recorded at any concentration tested.
The NOEC was determined to be 37 mg cation/kg soil dry weight. The EC50 could not be calculated, but it can be concluded that the EC50 is higher than 37 mg cation/kg soil dry weight.

Reported statistics and error estimates:

The statistical analysis was performed with the software. The Dunnett-test was 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 1 Effects of the test substance on growth (biomass increase during 4 weeks exposure) of adult earthworms.

Replicate	Initial fresh weight / worm (mg) (Mean per replicate)
1	357.8	364.0	363.8	364.9	367.1	362.1
2	370.4	371.6	380.7	368.9	372.2	376.7
3	387.2	389.9	381.8	384.6	381.9	388.1
4	404.8	390.8	392.4	392.9	392.2	392
5	352
6	379.4
7	382.5
8	393.7
Mean	378.5	379.1	379.7	377.8	378.4	379.7
SD	17.8	13.4	11.8	13.2	11.1	13.4
cv %	4.7	3.5	3.1	3.5	2.9	3.5
	Initial fresh weight / Worm (mg) after 4 weeks (Mean per replicate)
1	444.2	428	429.6	469.5	466.9	406.7
2	424.1	449.3	415	441.8	414.6	438.3
3	425.9	458.6	462.6	430.8	446.3	450.2
4	454.4	478.5	461.8	454.8	445.2	457.4
5	423.7
6	441
7	462.1
8	490.2
Mean	445.7	453.6	442.3	449.2	443.3	438.2
SD	23	21	23.8	16.7	21.6	22.4
cv %	5.2	4.6	5.4	3.7	4.9	5.1
	Biomass change (change in fresh weight after 4 weeks relative to initial fresh weight) weight / worm [mg] (mean per replicate)
1	86.4	64	65.8	104.6	99.8	44.6
2	53.7	77.7	34.3	72.9	42.4	61.6
3	38.7	68.7	80.8	46.2	64.4	62.1
4	49.6	87.7	69.4	61.9	53	65.4
5	71.7
6	61.6
7	79.6
8	96.5
Mean	67.2	74.5	62.6	71.4	64.9	58.4
SD	19.8	10.5	19.9	24.7	24.9	9.4
	Biomass change (change in fresh weight after 4 weeks relative to initial fresh weight) % (mean per replicate)
1	24.1	17.6	18.1	28.7	27.2	12.3
2	14.5	20.9	9	19.8	11.4	16.4
3	10	17.6	21.2	12	16.9	16
4	12.3	22.4	17.7	15.8	13.5	16.7
5	20.4
6	16.2
7	20.8
8	24.5
Mean	17.9	19.6	16.5	19	17.2	15.3

 SD: Standard deviation cv%: coefficient of variantion

Table 2.Effects of the test substance on mortality of adult earthworms and reproduction

Treatment group (mg cation/kg soil dw)	Conrtol	5	10	17	27	37
Replicate	Number of surviving adult worms per replicate (4 weeks after test initiation) Replicate
1	10	10	10	10	10	10
2	10	10	10	10	10	10
3	10	10	10	10	10	10
4	10	10	10	10	10	10
5	10
6	10
7	10
8	10
mean	10	10	10	10	10	10
SD	0	0	0	0	0	0
cv%	0	0	0	0	0	0
Mortality (%):
mean	0	0	0	0	0	0
Number of juvenile worms per replicate
1	98	129	127	96	135	89
2	113	104	117	101	122	118
3	86	83	103	110	108	120
4	116	112	67	132	75	96
5	92
6	135
7	124
8	104
mean	108.5	107	103.5	109.8	110	105.8
SD	16.6	19.1	26.2	15.9	25.8	15.6
cv%	15.3	17.8	25.4	14.5	23.5	14.7
Change of reproduction [%]
% to control	-	-1.4	-4.6	1.2	1.4	-2.5

 SD: Standard deviation cv%: coefficient of variantion

Calculation of key result

The original effect levels were expressed as cation species of the registered substance. The key effect levels are re-calculated and corrected to include the counterion species by multiplying with 1.868 (344.0 g/mol molecular weight of registered substance divided by 184.2 g/mol molecular weight of cation species).:

1.868 x 37 mg cation/kg soil dw = 69.1 mg registeretd substance/kg soil dw

Validity criteria fulfilled:

yes

Conclusions:

The NOEC was determined to be 37 mg cation/kg soil dry weight.

Executive summary:

A laboratory study was conduced to determine the chronic toxicity of the test substance to soil earthworm (Eisenia foetida). The study was based on OECD TG 222 and in compliance with GLP criteria. The study was performed with five test concentrations (5, 10, 17, 27 and 37 mg cation/kg soil) and a control group. Each test concentration had four replicates and the control had 8 replicates. Ten worms were used in each of the replicate. Thus, in total there were 40 organisms per test concentration and 80 organisms in the control. The test substrate was an artificial soil prepared with 69.6% industrial quartz sand, 20% kaolinite clay, 10% sphagnum peat and 0.4% calcium carbonate. There was34.4 - 35.2 % water content in the soil during the test.The test was conduced under 590 lux light intensity (16 hours on and 8 hours darkness), at a temperature of 18 - 21 °C and pH 5.9 – 6.3. The exposure period was 8 weeks (4 weeks to for adult mortality study and 4 weeks for juvenile development study). The behaviour and pathological symptoms of the earthworms (including feeding activity) were observed weekly, the survival of the adult and juveniles worms were recorded after 4 weeks exposure, and the morphological alterations of the juveniles were studied after 8 weeks exposure.

No mortality (0 %) occurred in the control group. The test item caused no mortality at any tested concentration after 4 weeks. No effects on behaviour (including feeding activity) of the worms were observed during the test.The test item caused no statistically significant change in biomass (change in fresh weight after 4 weeks relative to initial fresh weight) compared to the control group at all the concentrations tested, i.e. a weight increase of 17.9, 19.6, 16.5, 19.0, 17.2 and 15.3 % was recorded in the control group and at concentrations of 5, 10, 17, 27 and 37 mg cation/kg soil d.w., respectively.No statistically significant effects (Dunnet-Test, p > 0.05) on the number of juveniles compared to the control group were recorded at any concentration tested.

The EC50 could not be calculated, but it can be concluded that the EC50 is >37 mg cation/kg soil dry weight. The NOEC was determined to be 37 mg cation/kg soil dry weight.

 

 

Description of key information

14-d LC50 = 176.21 mg pure test substance/kg soil dw, mortality, Eisenia foetida, OECD TG 207, Bender 2006

56-d NOEC = 69.1 mg pure test substance/kg soil dw, reproduction, Eisenia foetida, OECD TG 222, Friedrich 2007

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:

176.21 mg/kg soil dw

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

69.1 mg/kg soil dw

Additional information

Short-term, 14-days

The acute toxicity of the test substance to soil earthworm (Eisenia foetida) was studied under GLP to OECD TG 207. A total of 40 organisms per concentration and control was exposed in an artificial soil prepared with 70% sand, 20% kaolin and 10% peat over a period of 14 days. A dose-dependent increase in mortality was seen in the other treatment groups, reaching a maximum of 90% at 295.98 and 587.19 mg cation/kg soil dw, respectively. The 14-day LC50 was determined to be 94.33 mg cation/kg of soil dw (equivalent to 176.21 mg pure test substance/kg soil dw).

Chronic, 56 days

The chronic toxicity of the test substance to soil earthworm (Eisenia foetida) was studied under GLP to OECD TG 222. A total of 40 organisms per test concentration and 80 organisms in the control (10 per replicate) were used. The test substrate was an artificial soil prepared with 69.6% industrial quartz sand, 20% kaolinite clay, 10% sphagnum peat and 0.4% calcium carbonate with 34.4-35.2% water content in the soil during the test. No mortality occurred in the control group. The test item caused no mortality at any tested concentration after 4 weeks. No effects on behaviour (including feeding activity) of the worms were observed during the test. The test item caused no statistically significant change in biomass (change in fresh weight after 4 weeks relative to initial fresh weight) compared to the control group. No statistically significant effects (Dunnett-Test, p > 0.05) on the number of juveniles compared to the control group were recorded at any concentration tested. The long-term NOEC was determined to be 37 mg cation/kg soil dry weight (equivalent to 69.1 mg pure test substance/kg soil dry weight).