4-(2-chlorophenyl)-N-cyclohexyl-N-ethyl-5-oxo-4,5-dihydro-1H-tetrazole-1-carboxamide

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:

supporting study

Study period:

28 Feb - 8 Apr 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Version / remarks:

version April 4, 1984

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Umwelt, Raumordnung und Landwirtschaft des Landes Nordrhein-Westfalen, Düsseldorf, Germany

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

- Method of mixing into soil (if used): For the main study, 50, 89, 158, 281, and 500 mg of the substance was weighed into finely ground sand to a total weight of 5000 mg each and mixed for ten minutes. At the same time, the rest of the deionised water required was mixed into the test soil in each test container. For the pre-study, 2000 mg of the test substance was weighed into 20 g finely ground sand and mixed thoroughly for ten minutes in a morter. The concentrations for the pre-study were prepared by mixing equivalent parts of this mixture into the test soil thoroughly with a domestic mixer.
- Controls: yes, test medium control

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: earthworm (reported as Eisenia fetida andrei)
- Source: strain of Prof. Graff, Forschungsanstalt für Landwirtschaft, Braunschweig, Germany. This strain has been held in the laboratory of Bayer AG for several years under the conditions described in the BBA Proposal for an OECD Guideline (ECO 85, = UPEC 15, "Test guideline for the assessment of toxicity to earthworms (Eisenia fetida Sav.), Laboratory test Draft", DBA AP-3000 b and 2600, February 1981).
- Age at test initiation (mean and range, SD): adult worms more than two months old
- Weight at test initiation (mean and range, SD): average weight was 0.28 g (average weight was 0.35 g in the pre-study)

ACCLIMATION
- Acclimation period: strain held for several years under constant conditions: 22 ± 2 °C, 70 - 90% relative humidity, 12:12 hours light-dark cycle. The substrate consists of about 70% by weight of natural soil, 25% peat and 5% straw (dry weight in each case). The animals are fed on ground, dried cattle manure at 14 day intervals. At the same time, the substrate is also replenished. On the day prior to the start of the study, they were removed from the breeding substrate for acclimatization and kept in the test soil (without test substance) under the test conditions until the start of the study.
- Acclimation conditions (same as test or not): yes, same as test
- Feeding: The animals were not fed during the study.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

14 d

Test temperature:

19 - 21 °C

pH:

6.2 - 6.3 (pH of test soil)

Moisture:

70 - 90% relative humidity

Details on test conditions:

TEST SYSTEM
- Test container (material, size): 1.5 liter preserving jars covered with glass lids
- Amount of soil or substrate: 500 g dry weight test soil (equivalent to 775 g wet weight)
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 4

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate): The test soil consists of 69% fine quartz sand (84% of the sand has a particle size of 0.06 - 0.2 mm), 10% dried, finely ground peat (sphagnum peat; pH 2 - 4), 20% kaolin (kaolinite content of about 36%, pH value ca. 7, "Kaolin W", from Erbslöh/Geisenheim) and around 1% calcium carbonate (pure) to adjust the pH value to 6 + 0.5 (figures refer to dry weight).
- Pretreatment of soil: The test soil was first of all mixed dry from these components in a plough-share mixer, and moistened with some of the final volume of water required.

OTHER TEST CONDITIONS
- Photoperiod: 12:12 hrs light-dark cycle
- Light intensity: 400 - 800 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Seven days after the start of the study, the number of surviving earthworms was counted by emptying the soil out onto an inert surface and removing the earthworms by hand. The animals were then returned to the test container with the test soil. After 14 days, the weight as well as the number of surviving earthworms were determined. Earthworms which show no reaction upon being prodded with a blunt probe are considered dead.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.78
- Range finding study: yes
- Test concentrations: control, 10, 100, and 1000 mg/kg dw (nominal)

Nominal and measured concentrations:

control, 100, 178, 316, 562, and 1000 mg/kg dw (nominal)

Reference substance (positive control):

yes

Remarks:

chloroacetamide

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:

mortality

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: weight alterations and symptoms

Duration:

14 d

Dose descriptor:

LOEC

Effect conc.:

178 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: weight alterations and symptoms

Details on results:

- Mortality at end of exposure period: none
- Total mass of adults at beginning of test: 6.79 g
- Changes in body weight of live adults (% of initial weight) at end of exposure period: see Table 3 below under "Any other information on results incl. tables"
- Behavioural abnormalities: none observed

Results with reference substance (positive control):

Under the same conditions, a study was carried out with the reference substance chloroacetamide A.R. on 20th November 1996 (Report HBF/Rg 238 of December 17, 1996) (test concentrations: 10, 18, 24, 32, and 56 mg/kg). The LC50 (14 days) determined in this study of 24 mg/kg dry weight soil (95 % confidence limits 23 - 25 mg/kg) is within the concentration range normally determined in international ring studies.

Reported statistics and error estimates:

The weight alterations of the test organisms were statistically evaluated by the U-Test of Wilcoxon, Mann & Whitney (L. SACHS: Angewandte Statistik, Springer Verlag 1978; Probability level P = 0.05, two sided). If possible, the EC50-values and the 95 percent confidence limits are calculated by an EC50 computer program developed by Dr. H.T. Ratte (Technical University Aachen) using the Probit-Analysis after the "Maximum-Likelihood" Method (according to Finney (1952)).

Table 1: Properties of the test soil (two determinations in each case) (No determinations of the maximum water capacity were made at the end of the study.)

Parameter	Pre-study				Main study
	Start of study		End of study		Start of study		End of study
pH value	6.2	6.2	6.2	6.1	6.2	6.2	6.2	6.3
water content in the test soil (%)	24.8*	25.7*	34.2	33.7	34.0*	33.6*	33.9	33.9
water content as % of the maximum water capacity	59.3	58.6	-	-	53.3	53.7	-	-

* The moisture content was determined in the soil pre-mix (plough-share mixer). When adding the test substance, 100 mL deionised water was also added to each test container so that the water content in the test soil was around 35% when the worms were introduced.

 

Table 2: Pre-study with Eisenia fetida to determine toxicity of the test substance to earthworm. Unless stated to the contrary, the figures in the table are means of n = 2 test containers, each containing ten earthworms.

Concentration (mg/kg dry weight soil)	Mortality after 14 days (%)	Weight alteration of the survivors (%)
Control	0	+ 4 ± 2
10	0	- 2 ± 0
100	0	- 2 ± 0
1000	0	- 20 ± 3

 

Table 3: Main study with Eisenia fetida to determine toxicity of the test substance to earthworm. Unless stated to the contrary, the figures in the table are means of n = 4 test containers, each containing ten earthworms.

Concentration (mg/kg dry weight soil)	Mortality after 14 days (%)	Weight alteration of the survivors (%)	U-Test*
Control	0	+ 9 ± 6
100	0	- 7 ± 4	-
178	0	- 1 ± 3	+
316	0	- 3 ± 6	+
562	0	- 8 ± 4	+
1000	0	- 16 ± 4	+

* Results of the U-test: + = weights of control and the test concentration do differ significantly (P = 0.05); - = weights of control and the test concentration do not differ significantly (P = 0.05)

 

Table 4: Individual figures obtained in the main study

Concentration (mg/kg dry weight soil)	Container number	Number of surviving worms			Mean weight of worms (g)
		Day 0	Day 7	Day 14	Day 0	Day 14
Control	1	10	10	10	0.28	0.30
	2	10	10	10	0.30	0.31
	3	10	10	10	0.27	0.32
	4	10	10	10	0.28	0.31
100	1	10	10	10	0.29	0.31
	2	10	10	10	0.28	0.29
	3	10	10	10	0.32	0.32
	4	10	10	10	0.27	0.31
178	1	10	10	10	0.31	0.32
	2	10	10	10	0.28	0.27
	3	10	10	10	0.29	0.28
	4	10	10	10	0.30	0.31
316	1	10	10	10	0.28	0.27
	2	10	10	10	0.27	0.27
	3	10	10	10	0.28	0.25
	4	10	10	10	0.28	0.29
562	1	10	10	10	0.29	0.27
	2	10	10	10	0.28	0.26
	3	10	10	10	0.29	0.25
	4	10	10	10	0.27	0.26
1000	1	10	10	10	0.27	0.24
	2	10	10	10	0.26	0.22
	3	10	10	10	0.29	0.24
	4	10	10	10	0.26	0.22

 

Table 5: Weight determinations of individual earthworms at the start of the study (main study)

Concentration (mg/kg dry weight soil)	Container number	Weight of worms (g) per individual worms
		1	2	3	4	5	6	7	8	9	10
Control	1	0.31	0.26	0.30	0.26	0.30	0.27	0.30	0.25	0.25	0.30
	2	0.30	0.31	0.32	0.26	0.39	0.31	0.25	0.25	0.36	0.29
	3	0.26	0.25	0.27	0.31	0.29	0.29	0.27	0.26	0.26	0.28
	4	0.25	0.26	0.26	0.34	0.25	0.30	0.38	0.25	0.26	0.25
100	1	0.29	0.28	0.27	0.26	0.35	0.27	0.25	0.42	0.25	0.28
	2	0.35	0.25	0.25	0.40	0.26	0.26	0.25	0.26	0.25	0.25
	3	0.52	0.26	0.34	0.28	0.25	0.25	0.30	0.25	0.35	0.25
	4	0.26	0.34	0.32	0.25	0.25	0.25	0.25	0.25	0.27	0.26
178	1	0.25	0.25	0.40	0.35	0.25	0.30	0.40	0.35	0.27	0.25
	2	0.30	0.30	0.25	0.27	0.25	0.27	0.32	0.25	0.31	0.29
	3	0.30	0.38	0.29	0.30	0.30	0.27	0.30	0.25	0.25	0.25
	4	0.50	0.33	0.31	0.31	0.25	0.31	0.25	0.28	0.25	0.25
316	1	0.33	0.25	0.32	0.26	0.26	0.28	0.32	0.26	0.29	0.25
	2	0.34	0.29	0.25	0.25	0.23	0.33	0.25	0.25	0.25	0.25
	3	0.30	0.25	0.30	0.27	0.26	0.28	0.25	0.26	0.30	0.33
	4	0.29	0.25	0.26	0.28	0.35	0.26	0.27	0.28	0.25	0.26
562	1	0.31	0.25	0.33	0.30	0.25	0.25	0.34	0.27	0.30	0.26
	2	0.34	0.34	0.25	0.25	0.25	0.25	0.25	0.33	0.28	0.25
	3	0.34	0.25	0.25	0.39	0.27	0.30	0.29	0.25	0.30	0.27
	4	0.28	0.32	0.27	0.32	0.27	0.24	0.25	0.26	0.25	0.25
1000	1	0.36	0.25	0.27	0.28	0.25	0.27	0.26	0.25	0.25	0.25
	2	0.25	0.32	0.29	0.25	0.25	0.26	0.25	0.25	0.25	0.27
	3	0.30	0.30	0.27	0.25	0.27	0.30	0.25	0.27	0.43	0.28
	4	0.31	0.28	0.25	0.25	0.27	0.25	0.25	0.25	0.25	0.25

Table 6: Validity criteria for OECD 207

Criterion from the guideline	Outcome	Validity criterion fulfilled
The mortality in the controls should not exceed 10 per cent at the end of test	0%	yes

 

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Reference 2

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Data waiving:

exposure considerations

Justification for data waiving:

the study does not need to be conducted because direct and indirect exposure of the soil compartment is unlikely

Additional information

In accordance with Regulation (EC) No 1907/2006, Annex IX, column 2, testing of effects on terrestrial organisms is not required since direct and indirect exposure of the soil compartment for the substance 4-(2-chlorophenyl)-N-cyclohexyl-N-ethyl-5-oxo-4,5-dihydro-1H-tetrazole-1-carboxamide is unlikely. Furthermore, the evaluation of the PEC/PNEC ratio indicates no risk for the terrestrial compartment (RCR << 1). For detailed information, see Chemical Safety Report (CSR) chapter 9 and 10.

One short-term study is available for soil macroorganisms , which was conducted according to GLP standards and followed OECD guideline 207. Earthworms (Eisenia fetida) were exposed to five concentrations (100, 178, 316, 562, and 1000 mg/kg dry weight soil) in an artificial soil consisting of sand, clay mineral and peat. The determined LC50 (14 d) was >1000 mg/kg dry weight soil (nominal). A NOEC (14 d) of 100 mg/kg dry weight soil (nominal) based on weight alterations was determined, the LOEC (14 d) was 178 mg/kg dry weight soil (nominal).