1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylindeno[5,6-c]pyran

Toxicity to soil macroorganisms except arthropods

Administrative data

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11/09/1996 - 22/08/1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Reliability 2 is assigned because the test has been carried out following internation guideling and under GLP without deviations that influence the results, however, the tested concentrations were not verified with expertimental analysis.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report Date:

1997

Materials and methods

Test guideline

Qualifier:

according to

Guideline:

ISO 11268-2 (Effects of Pollutants on Earthworms. 2. Determination of Effects on Reproduction)

Principles of method if other than guideline:

According to draft version of this guideline 1993 (OECD 207)

GLP compliance:

yes (incl. certificate)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

Batch # 7953-I

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
No data

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

Samples were taken at the beginning and the end of the experiment

Test substrate

Vehicle:

yes

Details on preparation and application of test substrate:

The test materials at appropriate concentrations were dissolved in equal amounts of acetone (solvent), mixed with the quartz sand and allowed to slowly evaporate and mixed with the standard soil containing 10% Sphagnum peat, 20% kaolinite clay, approximately 70% fine quartz-sand (grain size 0.1-0.5 mm) and 0.5% calcium carbonate to adjust to pH 6.0±0.5. After preparation of the test concentrations and an equilibrium period of one week, the test organisms were added to the soil. The test medium was not refreshed during the test period.

Test organisms

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

Adult worms with well developped clitellum of an age between 8 and 9 months (range finding test) and between 9 and 10 months(main test) were used. Prior to the test initiation they were acclimated in artificial soil under test conditions for a preiod of 4 days, fed from the same food source as in definitive test. They were added to the test system one week after addition of test substance. Weights between 340 and 540 mg but did not differ for more then 100 mg within this range in each test container. Fed weekly with cattle manure. Adults removed after 28 days, offspring remained up to 56 days.

Study design

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

8 wk

Remarks:

Adults removed after 4 wks, offspring remained in test containers for another 4 weeks

Post exposure observation period:

No data

Test conditions

Test temperature:

17 - 23 degrees Celsius.

pH:

At start of study pH 6.3 - 6.4
After 8 wks pH 6.2 - 6.3

Moisture:

water content adjusted to 40-60% of total water holding capacity, corresponding to approx. 30% of dry weight.
Water content at start of study: 32.7 - 34.3 % of dry weight, after 8 weeks: 37.0 - 40.9 % of dry weight

Details on test conditions:

Composition soil: 10% sphagnum-peat; 20% kaolinite clay;circa 0.5% CaCO3 to adjust pH to 6.0+/- 0.5; approx. 50.1% fine quartz sand (grain size 0.1 - 0.5 mm). Each test container contained 655 g soil (500 g dry soil + 150 g water (= 30%)) plus 5 g food. Food: 10 g finely ground cattle manure/kg soil + 5 g/kg at start on soil surface + weekly feeding of adults. Soil layer thickness circa 4 cm
DIN, solvent: acetone, test range: 8 - 250 mg/kg
16 h light/8h dark, 420 - 740 lux.

Nominal and measured concentrations:

Nominal concentrations were 8, 19, 45, 105 and 250 mg/kg soil dw. Samples were taken in case analyses would be considered to be necessary. No analyses were carried out.

Reference substance (positive control):

yes

Remarks:

Benomyl, 50% active ingredient, 15 mg/kg soil (dw)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

8 wk

Dose descriptor:

NOEC

Effect conc.:

45 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality, growth, reproduction

Effect concentrations 2

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

105 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality, growth, reproduction

Effect concentrations 3

Duration:

4 wk

Dose descriptor:

NOEC

Effect conc.:

105

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Effect concentrations 4

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

250 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: reproduction and food consumption

Effect concentrations 5

Duration:

4 wk

Dose descriptor:

NOEC

Effect conc.:

>= 250 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

Mortality of the adults was not affected after 4 weeks in concentrations up to 250 mg/kg. In the range finding test mortality was 100% after 14 days exposure to 1000 mg/kg. Growth was significantly inhibited (15%) in the highest concentration of 250 mg/kg. Reproduction was not significantly affected up to concentrations of 45 mg/kg (7% inhibition, NOEC). At the level of the LOEC (105 mg/kg), the reproduction was 57% of the control, whereas in the highest concentration reproduction was inhibited completely. Reduction in the food consumpation was observed at a concentration of 250 mg/kg.

Results with reference substance (positive control):

Results with 15 mg benomyl/kg after 4 weeks showed no mortality and 12% inhibition of growth as compared to the controls. The reproduction was almost completely inhibited.

Reported statistics and error estimates:

For each concentration a statistical analysis of homogeneity and normality of replicate results was made. Because no significant difference (Dunnett test, p=0.05) between the water treated control and the solvent control was observed, both controls were pooled. Appropriate statistical analysis was performed to determine whether differences between the pooled control and the test concentrations were significant (Dunnett test, p = 0.05)

Any other information on results incl. tables

 

Table - Mortality after 4 weeks per replicate (mean per treatment±standard deviation, n = 4)

Treatment	Concentration (mg/kgdw)	Mean (% of initial number))	Std.dev. (%)
control	0	0.0	0.0
solvent control	0
C1	8.0	0.0	0.0
C2	19.0	0.0	0.0
C3	45.0	0.0	0.0
C4	105.0	0.0	0.0
C5	250.0	0.0	0.0
reference compound		0.0	0.0

 

Table – Body weight changes after 4 weeks per replicate (mean per treatment±standard deviation, n = 4)

Treatment	Concentration (mg/kgdw)	Mean (body weight differences) in mg/worm	Std.dev.
control	0	20	35
solvent control	0
C1	8.0	18	22
C2	19.0	58	28
C3	45.0	11	44
C4	105.0	84	85
C5	250.0	-66	23
reference compound		-45	25

 

Table – Reproduction after 8 weeks per replicate (mean per treatment±standard deviation, n = 4)

Treatment	Concentration (mg/kgdw)	Mean ((number of young worms)	Std.dev.
control	0	241	25
solvent control	0
C1	8.0	228	37
C2	19.0	237	20
C3	45.0	224	15
C4	105.0	138	38
C5	250.0	0	0
reference compound		3	3

 

Table – Food consumption over weeks 1 - 5 per replicate (mean per treatment±standard deviation, n = 4)

Treatment	Concentration (mg/kgdw)	Mean added food (g)	Std.dev.
control	0	21.2	0.7
solvent control	0
C1	8.0	21.5	0.5
C2	19.0	22.4	0.4
C3	45.0	21.8	0.3
C4	105.0	20.2	1.3
C5	250.0	10.9	0.5
reference compound		16.2	1.4

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

mortality in controls after 4 wks was 0 % (required < 10%); reproduction in each replicate was more then 200 (required more then 30); coeff. of variation for reproduction was circa 10% (required less then 30%).

Conclusions:

8wk-NOEC (reprod., survival) = 45 mg/kg DW; 4wk-NOECgrowth = 105 mg/kg DW; 4wk-NOECsurvival >=250 mg/kg DW.

Executive summary:

The earthworm Eisenia foetida was tested. The test materials at appropriate concentrations were dissolved in equal amounts of acetone, mixed with the quartz sand and allowed to slowly evaporate and mixed with the standard soil  containing 10% Sphagnum peat, 20% kaolinite clay, approximately 70% fine quartz-sand (grain size 0.1-0.5 mm) and 0.5% calcium carbonate to adjust to pH 6.0±0.5. After preparation of the test concentrations and an equilibrium period of one week, the test organisms were added to the soil. The test medium was not refreshed during the test period.The earthworm test was carried out according to ISO 11268. Adult worms (Eisenia fetida) were exposed to nominal concentrations in soil of 8, 19, 45, 105 and 250 mg/kg. Weights of the adult worms ranged between 340 and 540 mg, but did not differ more than 100 mg within this range in each test container. The worms were fed weekly with finely ground cattle manure. Adult worms were removed after 4 weeks of exposure, counted and weighed. The remaining offspring remained in the test containers for another four weeks. Mortality of the adults was not affected after 4 weeks in concentrations up to 250 mg/kg. In the range finding test mortality was 100% after 14 days exposure to 1000 mg/kg. Growth was significantly inhibited (15%) in the highest concentration of 250 mg/kg. Reproduction was not significantly affected up to concentrations of 45 mg/kg (7% inhibition, NOEC). At the level of the LOEC (105 mg/kg), the reproduction was 57% of the control, whereas in the highest concentration reproduction was inhibited completely.