Reaction mass of 2-(3,4-dimethyl-1H-pyrazol-1-yl)succinic acid and 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinic acid

Toxicity to terrestrial arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to terrestrial arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 214 (Honeybees, Acute Contact Toxicity Test)

Version / remarks:

1998

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatsministerium für Umwelt und Landwirtschaft Freistaat Sachsen, Germany

Application method:

contact

Analytical monitoring:

no

Vehicle:

yes

Details on preparation and application of test substrate:

- Method of test material application: single droplet
- Body part: dorsal bee thorax
- Volume of test solution applied: 2 µL
- Controls: deionised water; Deionised water with 1 % wetting agent; positive control: Dimethoate EC 400
- Name of vehicle (organic solvent, emulsifier or dispersant): Tween80, active ingredient: 80 g/L Polyoxyethylene sorbitan monooleate
- Concentration of vehicle in test medium (stock solution and final test solution): 1.0 % (v/v)

Test organisms (species):

Apis mellifera

Animal group:

Hymenoptera (honeybees)

Details on test organisms:

TEST ORGANISM
- Common name: worker honeybees
- Source: Bienenfarm Kern GmbH, Am Rehbacher Anger 10, 04249 Leipzig-Rehbach, Germany
- Age at test initiation (mean and range, SD): The approximate age of female bees used in the test depends on physiological, ecological and behavioural factors during holding of the bees, but was generally between 3 and 5 weeks as typical for “summer” bees.
- Date of collection: collected in the morning of use and kept under test conditions
- Cultural background (if honeybees): worker honeybees
- Disease free: yes / no
- Kept according to standard practices: yes; from healthy, disease free and queen-right bee colony

Study type:

laboratory study

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

24.7 – 25.2 °C

Humidity:

58 - 62 %

Photoperiod and lighting:

constant darkness throughout the test (diffuse artificial light of about 100 lx only during handling and assessments)

Details on test conditions:

TEST SYSTEM
- Test container / cage (material, size): Climatic chamber, cages of cardboard with holes in the bottom for ventilation and a glass plate in front, 95 mm x 50 mm x 65 mm (length x width x height)
- No. of organisms per container: 10
- No. of replicates (containers) per treatment: 6
- No. of replicates (containers) per control: 6

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Mortality: number of dead bees after 4, 24 and 48 hours
Behaviour: The number of bees showing normal behaviour and the number of bees showing any behavioural abnormalities after 4, 24 and 48 hours e.g health, effectiveness (paralysis, lateral position, lying on the back, abnormal movements), any differences in activity, in position within the cage (e.g. all on the bottom) or any abnormality in amount and colour of excretion were documented. All observations were made in comparison to the control bees.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 201.4, 100.7 and 50.4 μg A 1/bee
- Results used to determine the conditions for the definitive study: no significant mortality occurred

Nominal and measured concentrations:

Nominal: 100.7 µg product /bee; 100.0 µg a.s./bee

Reference substance (positive control):

yes

Remarks:

Dimethoate EC 400 (BAS 152 11 l)

Duration:

48 h

Dose descriptor:

LD50

Effect conc.:

> 100.7 µg per animal

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

48 h

Dose descriptor:

LD50

Effect conc.:

> 100 µg per animal

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Details on results:

- Mortality: No mortality on honeybees after 48 hours was observed in controls (deionised water or tween80® solution) or in the treatment group
- Behavioural abnormalities: none

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: LD50 (48 h)= 0.135 μg dimethoate/bee (95 %-CL / lower-upper: 0.127 – 0.144 μg dimethoate/bee)

Reported statistics and error estimates:

Statistical program used: ToxRat Professional 2.10.06 (2010)
LD50: no LD50 calculation
- Probit ananlysis (linear maximum likelihood regression)
Statistical significance of mortality values (one-sided greater)
- Fisher’s Exact Binomial Test (α ≤ 0.05)
- Fisher’s Exact Binomial Test with Bonferroni Correction (α ≤ 0.05)

Validity criteria fulfilled:

yes

Reference 2

Endpoint:

toxicity to terrestrial arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 213 (Honeybees, Acute Oral Toxicity Test)

Version / remarks:

1998

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatsministerium für Umwelt und Landwirtschaft Freistaat Sachsen, Germany

Application method:

oral

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

- Details of food source: 50 % (w/v) aqueous sucrose solution
- Method of feeding during study: Food was offered continuously, using a glass feeding tube which was placed on the floor in the test cage.

Test organisms (species):

Apis mellifera

Animal group:

Hymenoptera (honeybees)

Details on test organisms:

TEST ORGANISM
- Common name: worker honeybees
- Source: Bienenfarm Kern GmbH, Am Rehbacher Anger 10, 04249 Leipzig-Rehbach, Germany
- Age at test initiation (mean and range, SD): The approximate age of female bees used in the test depends on physiological, ecological and behavioural factors during holding of the bees, but was generally between 3 and 5 weeks as typical for “summer” bees.
- Date of collection: collected in the morning of use and kept under test conditions
- Cultural background (if honeybees): worker honeybees
- Disease free: yes / no
- Kept according to standard practices: yes; from healthy, disease free and queen-right bee colony

Study type:

laboratory study

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

24.7 – 25.2 °C

Humidity:

58 - 62 %

Photoperiod and lighting:

constant darkness throughout the test (diffuse artificial light of about 100 lx only during handling and assessments)

Details on test conditions:

TEST SYSTEM
- Test container / cage (material, size): Climatic chamber, cages of cardboard with holes in the bottom for ventilation and a glass plate in front, 95 mm x 50 mm x 65 mm (length x width x height)
- No. of organisms per container: 10
- No. of replicates (containers) per treatment: 6
- No. of replicates (containers) per control: 6

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Mortality: number of dead bees after 4, 24 and 48 hours
Behaviour: The number of bees showing normal behaviour and the number of bees showing any behavioural abnormalities after 4, 24 and 48 hours e.g health, effectiveness (paralysis, lateral position, lying on the back, abnormal movements), any differences in activity, in position within the cage (e.g. all on the bottom) or any abnormality in amount and colour of excretion were documented. All observations were made in comparison to the control bees.

FOOD CONSUMPTION (if oral study)
- Amount of treated diet consumed per group (10 bees): control: 236.100 g; test group: 232.100 g

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 201.4, 100.7 and 50.4 μg A 1/bee
- Results used to determine the conditions for the definitive study: no significant mortality occurred

Nominal and measured concentrations:

Nominal: 120.8 μg A 1/bee; equivalent to 120.0 μg a.s./bee
Consumed: 119.2 μg A 1/bee; equivalent to 118.4 μg a.s./bee
(The unit “μg a.s./bee” refers to the analysed purity of A 1)

Reference substance (positive control):

yes

Remarks:

Dimethoate EC 400 (BAS 152 11 l)

Duration:

48 h

Dose descriptor:

LD50

Effect conc.:

> 119.2 µg per animal

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: consumed

Duration:

48 h

Dose descriptor:

LD50

Effect conc.:

> 118.4 µg per animal

Nominal / measured:

estimated

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: consumed

Details on results:

- Mortality: No statistically significant effects on mortality of honeybees occurred at the tested dose rate.
- Behavioural abnormalities: none

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: LD50 (48 h)= 0.097 μg/bee (95 %-CL / lower-upper: 0.088 – 0.107 µg/bee)

Reported statistics and error estimates:

Statistical program used: ToxRat Professional 2.10.06 (2010)
LD50: no LD50 calculation
- Probit ananlysis (linear maximum likelihood regression)
Statistical significance of mortality values (one-sided greater)
- Fisher’s Exact Binomial Test (α ≤ 0.05)
- Fisher’s Exact Binomial Test with Bonferroni Correction (α ≤ 0.05)

Validity criteria fulfilled:

yes

Reference 3

Endpoint:

toxicity to terrestrial arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 Jul - 28 Aug 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

ISO 11267 (Inhibition of Reproduction of Collembola by Soil Pollutants)

Version / remarks:

1999

Qualifier:

according to guideline

Guideline:

OECD Guideline 232 (Collembolan Reproduction Test in Soil)

Version / remarks:

2009

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatsministerium für Umwelt und Landwirtschaft Freistaat Sachsen, Germany

Application method:

soil

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

- Method of mixing into soil: An exactly weighed amount of the test item was dissolved in deionised water to make a stock solution, without addition of solubility mediators, immediately before application. This stock solution was diluted with deionised water such that each solution contained the amount of test item in 25 mL required to dose 250 g soil dry weight (at 15 % dry weight water content). Each test item solution (25 mL) was thoroughly mixed with the artificial soil using a laboratory mixer.
- Controls: control soil treated with deionised water only
- Volume of test solution applied: 25 mL

Test organisms (species):

Folsomia candida

Animal group:

Collembola (soil-dwelling springtail)

Details on test organisms:

TEST ORGANISM
- Common name: Folsomia candida (Willem)
- Source: originally purchased from “Biologische Bundesanstalt (BBA)”, Berlin-Dahlem in May 2000; reared in the laboratory of the test facility under ambient laboratory conditions
- Age at test initiation (mean and range, SD): 9 - 12 days (juvenile collembolans)
- Breeding medium: mixture of plaster of Paris, activated charcoal and water (8:1:9)
- Breeding conditions: breeding container: plastic vessel of Bellaplast with a transparent lid (9.5 cm x 9.5 cm x 6 cm); moistening automatically by means of an absorbent wick aeration in combination with feeding; food source: granulated dry yeast, supplied twice a week; light : dark cycle = 16 h : 8 h (diffuse artificial light); temperature: nominally 20 ± 2 °C

Study type:

laboratory study

Limit test:

no

Total exposure duration:

28 d

Test temperature:

18.0 – 21.9 °C

pH (if soil or dung study):

test initiation: 5.91 - 6.08
test completion: 5.72 - 5.81

Humidity:

Max. water holding capacity WHC (g/100 g soil d.w.): 42.9

Photoperiod and lighting:

source: artificial light (LumiLux L58W)
intensity: 510 lx
duration: light : dark = 16 h : 8 h

Details on test conditions:

TEST SYSTEM
- Test container (material, size): glass container (approximately 150 mL) covered with a glass lid
- Amount of soil or substrate: 250 g
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4 ( + 2 replicates not loaded with collembolans for measurement purposes)
- No. of replicates per control: 8 ( + 2 replicates not loaded with collembolans for measurement purposes)

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition of artificial soil:
- 5 % sphagnum peat; origin: Torfwerk Moorkultur Ramsloh,
26683 Saterland, Germany, classified according to DIN 11540 (as close to pH 5.5-6.0 as possible, no visible plant parts remained,
finely ground, dried to measured moisture content)
- 20 % kaolin clay (kaolinite content > 30 %);
type: Kaolin W, origin: ERBSLÖH Lohrheim GmbH, 65558 Lohrheim, Germany
- 0.3 % calcium carbonate; origin: MERCK KGaA, 64271 Darmstadt, Germany
- 74.7 % industrial quartz sand; type: Millisil W3,
origin: Quarzwerke GmbH, 50207 Frechen, Germany
(fine sand is dominant with more than 50 % of the particles between 50 and 200 μm)
- deionised water

OTHER TEST CONDITIONS
- Photoperiod: light : dark = 16 h : 8 h
- Light intensity: 510 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
adult mortality and reproduction rate after 28 days

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8

Nominal and measured concentrations:

Nominal: control, 16, 29, 53, 95, 171, 309, 556 and 1000 mg test item/kg soil d.w.

Reference substance (positive control):

yes

Remarks:

Boric acid

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

mortality

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

mortality

Details on results:

- No significant effect was observed at any of the tested concentrations
- Mortality at end of exposure period: <= 5%
- No. of offspring produced: see Any other information on results incl. tables

Results with reference substance (positive control):

To verify the sensitivity of the test system the reference item boric acid is routinely tested at concentrations of 44, 67, 100, 150 and 225 mg/kg soil dry weight. The collembolans of the reference test were from the same source culture as those used in the definitive test. In the most recent study (BioChem project No. R 13 10 48 004 S, dated 16 July 2013) the EC50 was determined to be 108 mg/kg soil dry weight. The LC50 was determined to be 192 mg/kg soil dry weight. The NOEC for mortality and for reproduction was determined to be 100 and 44 mg/kg soil dry weight, respectively.

Reported statistics and error estimates:

The statistical analysis was performed with the software ToxRat Professional 2.10.06 (Ratte 2010). The Shapiro-Wilk´s test and Levene´s test, respectively, were used to test the data for normality and homogeneity of variance. 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.

Table: Surviving parental collembolans

Treatment group	mg test item/kg soil d.w.
	Control	16	29	53	95	171	309	556	1000
Mean	9.5	9.5	9.5	9.8	9.5	10.0	9.5	9.5	9.5
SD	0.8	0.6	0.6	0.5	0.6	0.0	0.6	0.6	0.6
Mortality (%)	5.0	5.0	5.0	2.5	5.0	0.0	5.0	5.0	5.0

Table: Number of juveniles

Treatment group	mg test item/kg soil d.w.
	Control	16	29	53	95	171	309	556	1000
Mean	754	764	736	777	753	734	746	744	724
SD	66.4	55.6	71.2	124.2	71.9	61.4	29.0	52.6	42.7
% Reduction of reproduction compared to control	-	-1	2	-3	0	3	1	1	4

Validity criteria fulfilled:

yes

Description of key information

NOEC (28 d) >= 1000 mg/kg soil d.w. (Folsomia candida; OECD 232)

LD50 (48 h) > 100 µg a.i./bee (Apis mellifera; OECD 214)

LD50 (48 h) > 118.4 µg a.i./bee (Apis mellifera; OECD 213)

Additional information

Two studies are available on the toxicity of Reaction mass of 2-(3,4-dimethyl-1H-pyrazol-1-yl)succinic acid and 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinic acid to terrestrial arthropods.

The long-term study conducted with Folsomia candida was performed according to OECD Guideline 232 and GLP (Friedrich, 2013). The test organism was exposed to the test substance at nominal concentrations of 16, 29, 53, 95, 171, 309, 556 and 1000 mg test item/kg soil d.w. for 28 days. No significant effect was observed at any of the tested concentrations, and the NOEC is thus determined to be >= 1000 mg/kg soil d.w..

Toxicity to the honeybee Apis mellifera was tested in a short-term study with oral and contact exposure, according to the OECD Guidelines 213 and 214, respectively (Franke, 2013). In the oral study bees were fed with a 50 % (w/v) aqueous sucrose solution containing the test substance. The consumption of test substance was estimated to 118.4 μg a.i./bee during the 48 h test period. As no significant mortality or behavioural abnormalities occurred, the LD50 for oral exposure was determined to be > 118.4 μg a.i./bee. For the contact toxicity test, the test substance was dissolved in deionised water with 1 % Tween80. A single droplet of this solution was applied on the dorsal thorax of the bees, resulting into a dose of 100 µg a.i. /bee. No mortality or behavioural abnormalities were observed during the 48-hour test period, and the LD50 for contact toxicity was determined to be >100 µg a.i. /bee.