potassium (oxido-NNO-azoxy)cyclohexane; cyclohexylhydroxydiazene 1-oxide, potassium salt; [K-HDO]

Toxicity to soil macroorganisms except arthropods

Administrative data

Endpoint:

toxicity to soil macroorganisms except 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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Cyclohexylhydroxydiazene 1-oxide, potassium salt

EC Number:

613-953-8

Cas Number:

66603-10-9

Molecular formula:

C6H11KN2O2

IUPAC Name:

Cyclohexylhydroxydiazene 1-oxide, potassium salt

Test material form:

liquid

Specific details on test material used for the study:

Name: Xyligen LP 15684
Batch No.: VM 2012
Received: 27.09.2004
Expiration date: 28.07.2006
Formulation type: aqueous solution
Chemical name: N-Cyclohexyldiazeniumdioxy-potassium
Purity: 30 % (30.41 % analysed)
Appearance: yellowish liquid
Density: 1.12 g/cm³ at 25 °C
Solubility in water: Soluble
Storage conditions: room temperature (> 15 °C)
Stability: expected to be stable for at least for 2 years

Sampling and analysis

Analytical monitoring:

no

Test substrate

Vehicle:

yes

Remarks:

water

Details on preparation and application of test substrate:

Test solutions were made by dispersing weighed amounts of the test item in deionised water, immediately prior to application. The test item was dispersed in sufficient deionised water such that the addition of the test solutions to the basic substrate (at 25 % water content) resulted in a final water content of the substrate of 35 %. Treated substrate was thoroughly mixed using a mixing machine immediately before application.

Test organisms

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Eisenia fetida (SAVIGNY) 1926, subspecies Eisenia fetida andrei (BOUCHÉ)
- Source: own breed of the test laboratory; originally purchased from "W. Neudorff GmbH KG", An der Mühle 3, D-31860 Emmerthal
- Age at test initiation: about 6 months old with clitellum
- Weight at test initiation (mean and range, SD): 364.3 - 439.2 mg/worm;
mean values/replicate:
Replicate 1 2 3 4
Mean 403.6 402.4 403.3 402.2
SD 30.9 22.6 14.9 13.8
cv % 7.7 5.6 3.7 3.4

ACCLIMATION
- Acclimation period: approximately 24 hours
- Acclimation conditions (same as test or not): same
- Health during acclimation (any mortality observed): no mortality observed

Study design

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

14 d

Test conditions

Test temperature:

18 - 21 °C

pH:

pH= 5.9 - 6.0 (beginning) and 5.8 - 5.9 (test termination)

Moisture:

final nominal moisture content of 35 % dry weight

Details on test conditions:

TEST SYSTEM
- Test container (material, size): clear glass container with clear glass lid (volume: 1 litre)
- Amount/test vessel: about 750 g wet weight corresponding to 556 g dry weight of artificial soil with about 35 % water content
- No. of organisms per container (treatment): 10
- Number of animals/vessel (= replicate): 10
- Number of replicates/treatment group: 4
- Number of animals/treatment group: 40

PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate):
10 % sphagnum peat
20 % kaolinite clay
0.5 % calcium carbonate
69.5 % industrial quartz sand (more than 50 % of the particles between 0.05 mm and 0.2 mm)
deionised water
- Maximum water holding capacity (in % dry weigth): 66.5 %
- Pretreatment of soil: About 24 hours before starting the test, the artificial soil was prepared for use. Deionised water was added to the dry components to give a moisture content of about 25 % of the dry weight.
- Stability and homogeneity of test material in the medium: stable

OTHER TEST CONDITIONS
- Photoperiod: continuous lighting (fluorescent light)
- Light intensity: 600 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
At the start of the test:
- Fresh weight per worm (mg/worm) by weighing each worm separately
- Behaviour of earthworms (reactivity to light and touch, burrowing time)
- Physico-chemical parameters of the artificial soil (water content, pH) (analysed from pooled samples of each treatment group separately)

7 days after application:
- Number of surviving earthworms for each test vessel
- Condition and behaviour of earthworms (reactivity to light and touch)
- Morphological alterations (fluid filled herniations, blisters, ulcers, twisted or segregated segments)

14 days after application:
- Number of surviving earthworms for each test vessel
- Condition and behaviour of earthworms (reactivity to light and touch)
- Morphological alterations (fluid filled herniations, blisters, ulcers, twisted or segregated segments)
- Fresh weight per surviving earthworm (mg/worm) by weighing each worm separately
- Physico-chemical parameters of the artificial soil (water content, pH) (analysed from pooled samples of each treatment group separately

TEST CONCENTRATIONS
- Test concentrations: 100, 500 and 1000 mg test item/kg soil dry weight (d.w.)

Reference substance (positive control):

yes

Remarks:

2-chloroacetamide

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Biomass decrease

Effect concentrations 2

Key result

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:

other: No mortality, abnormal behaviour or pathological symptoms of the worms were observed in any treatment or control group during the test.

Details on results:

- Mortality at end of exposure period: 0%
- Changes in body weight of live adults (% of initial weight) at end of exposure period: Mean fresh weights of surviving worms were reduced by 3.3 % in the control and 3.4, 5.0 and 7.4 % for the 100, 500 and 1000 mg Xyligen LP 15684/kg soil d.w. treated groups, respectively. The weight reduction was statistically significant (p ≤ 0.05) relative to the control at the test item concentrations of 500 and 1000 mg Xyligen LP 15684/kg soil d.w., respectively.
- Morphological abnormalities: 0 %
- Behavioural abnormalities: 0 %
- Other biological observations: none observed

Results with reference substance (positive control):

To verify the sensitivity of the test system, the reference item 2-chloroacetamide was routinely tested at concentrations of 14.1, 18.3, 23.8, 31.0 and 40.4 mg a.i./kg soil dry weight. In the most recent test, dated 07.07.2004 (BioChem project No.: TEA-R 04 10 48 002), the 14-day LC50 was calculated as 29.98 mg a.i./kg soil dry weight.

Reported statistics and error estimates:

The endpoints were mortality and fresh weight of surviving worms after 14 days. The arithmetic mean and the standard deviation per treatment and control for biomass were calculated.
The statistical analysis was performed with the software ToxRat Professional 2.07 (RATTE 2002). The DUNNETT-test was used to compare the control with the independent test item groups and showed a non-significant or a significant difference between both.
For statistical evaluation of the body weight change, the changed mean fresh weight of surviving worms per replicate was used.

Any other information on results incl. tables

Summary of test results:

	Test concentration Xyligen LP 15684 (mg/kg artificial soil)
Test parameter	0 (control)	100	500	1000
Mortality (%)	0	0	0	0
Abnormal behaviour, or pathological symptoms	None	None	None	None
Biomass decrease (mean %)	3.3	3.4	5.0	7.4
Temperature (°C)	18 - 21 °C
pH	5.8 – 6.0
water content	35.1 – 35. 3 % at the beginning and 34.6 – 35.3 % at test termination corresponding to 52.8 – 53.1 % and 52.0 – 53.1 % of water holding capacity

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

The validity criterion for the control group was accomplished: adult mortality: ≤ 10 % (being 0 % after 14 days)

Conclusions:

In a 14-day toxicity study with Xyligen LP 15684 (K-HDO 30 % in water) on earthworm (Eisenia fetida) the LC50 was determined to be greater than 1000 mg Xyligen LP 15684/kg soil dry weight. The NOEC was determined to be 100 mg Xyligen LP 15684/kg soil dry weight.

Executive summary:

The study was performed according to OECD 207 (1984). The artificial soil filled in glass vessels was treated with different concentrations of the test item before earthworms were placed on top of the soil. Four treatment groups (three test item concentrations, one water treated control) with four replicates each and each containing ten adult earthworms. The number of surviving adult earthworms and their weight loss after 14 days as well as behaviour and pathological symptoms were determined.

The test item caused no mortality at any tested concentrations. No mortality occurred in the control group. No abnormal behaviour or toxic symptoms of the worms were observed in any treatment or control group during the test.

Mean fresh weights of surviving worms were reduced by 3.3 % in the control and 3.4, 5.0 and 7.4 % for the 100, 500 and 1000 mg test item/kg soil d.w. treated groups, respectively. The weight reduction was statistically significant (p ≤ 0.05) relative to the control at the test item concentrations of 500 and 1000 mg test item/kg soil d.w., respectively.

Based on the statistical evaluation of these results, the no-observed-effect-concentration (NOEC) was determined at 100 mg test item/kg soil dry weight.

The LC₅₀could not be calculated but it can be concluded that the LC₅₀is greater than 1000 mg/test item/kg soil dry weight.