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Toxicity to soil macroorganisms except arthropods

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Reference
Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006-01-24 to 2006-02-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD 207 guideline with all validity criteria fulfilled and including a GLP compliance statement.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 207 (Earthworm, Acute Toxicity Tests)
Deviations:
yes
Remarks:
LC50 was not calculated as the induced mortality exhibited a sudden increase from 2.5% up to 92.5% for the 499 and the 799 mg/kg dw treated groups respectively. Based on the regularity of the results, 499 < LC50< 799 mg/kg dry soil was estimated.
GLP compliance:
yes (incl. certificate)
Remarks:
2006-04-18
Analytical monitoring:
no
Details on sampling:
not apllicable
Vehicle:
yes
Details on preparation and application of test substrate:
Test substance: For both the preliminary test and the full test, stock solutions were previously prepared in acetone. The concentration of the solutions was calculated so that the treatment concentrations were achieved by adding 2.0 mL of solution to 10 g of sand. Acetone was then allowed to evaporate and the coated sand was mixed with the soil portion.
Solvent control: The solvent control units were treated with an equivalent volume of sand previously added with acetone only. Acetone was evaporated and the sand was mixed with the soil fractions.
Water control: The water control soil received untreated sand.
For the test substances treated units, the solvent control and the water control units, watering of soil was performed after the treatment applications and the soil portions were firmly mixed prior the introduction of the specimens.
Reference substance (Chloroacetamid): water was used as the carrier. The substance was diluted in the volume of water needed to moisten the soil up to 50% of its total water holding capacity. For that purpose, a 25 g/L stock solution was prepared using about 0.5 g of substance in 20 mL of water. Aliquots of 0.4; 0.8 and 1.6 mL were added to the volume of water needed to moisten the soil so as to achieve final treatment concentrations of 20. 40 and 80 mg/kg dry soil.
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: Earthworm
- Source: adult specimens born at the Phytosafe site
- Age at test initiation (mean and range, SD): same generation between 2 and 12 months
- Weight at test initiation (mean and range, SD): 434-501 mg in the preliminary test and 441-573 mg in the final test.

ACCLIMATION
- Acclimation period: no data
- Acclimation conditions (same as test or not): the specimens involved in the tests were previously weighted in the breeding chamber and distributed 10 by 10 in glass vials, ready for their incorporation in the test units.
- Health during acclimation (any mortality observed): no data
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
14 d
Remarks:
for both preliminary and final tests
Post exposure observation period:
None
Test temperature:
The temperature was continuously controlled using a calibrated thermo-recorder and was 18.5-20.5 °C during the range-finding test and 20.5-22 °C in the final test.
pH:
pH of the soil substrate was measured at the beginning and the end of the exposure period and on day 7 for each unit where percent mortality was 100% on this day. It ranges from 5.6-6.1 during the preliminary study and from 5.6-6.2 during the full test.
Moisture:
Moisture of the soil substrate was controlled to range between 46.8 and 48.8 % of the total water holding capacity at the end of the final test.
Details on test conditions:
TEST SYSTEM
- Test container (material, size): 1.5 to 2.0 L capacity glass containers
- Amount of soil or substrate: 500 g equivalent dry soil substrate
- No. of organisms per container (treatment): 10 earthworms per unit
- No. of replicates per treatment group: 1 and 4 per treatment concentration for the range-finding test and the final test respectively.
- No. of replicates per control: 1 and 4 per treatment concentration for the range-finding test and the final test respectively.
- No. of replicates per vehicle control: 1 and 4 per treatment concentration for the range-finding test and the final test respectively.
The test units were covered by a plastic mesh in order to avoid escaping while allowing exchange of air.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
The composition of the soil substrate (dry w/w) was as follows:
-Sphagnum peat (grouned and sieved) 10%
- 100% Kaolinite clay 20%
- Quartz sand 150-210 µm 70%
1.8 g Calcium carbonate was added in each container that was previously calculated to allow a pH value of 6.0 +/- 0.5 on the control soil basis. Few days before the test, the sphagnum peat was grounded and sieved. Mean moisture was assessed from weight losses of 3 samples after drying. Total water holding capacity of each material was measured according to the procedure in ISO 11274 (1992). The results served for the calculation of total water holding capacity of the artificial soil, 100%-WHCsoil expressed as grams of water per 100 g dry weight of soil.

OTHER TEST CONDITIONS
- Photoperiod: continuous light
- Light intensity: within 400 and 800 lux controlled on days 0; 7 and 14 of the exposure period.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Induced mortality (at days 7 and 14 and any abnormal behaviour was reported); Mean weight of specimens (at the end of the exposure period, the survivors were rinsed and dried and mean weight was calculated as total weight reported to the number of survivors); pH of the soil substrate (at the beginning and the end of the exposure period and on day 7 for each unit where percent mortality was 100% on this day); Soil moisture (the control soil was sampled at the end of the test period and dried at 50°C toconstant weight and corresponding percent of the total water holding capacity was calculated).

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study: see Table 1
- Test concentrations: see Table 1
Nominal and measured concentrations:
Nominal concentrations:
- Range-finding test: 0.1; 2.0; 10.0; 100.1 and 1001.0 per kg dry soil.
- Full test: 121.1; 194.1; 311.1; 498.5 and 798.9 mg per kg dry soil.
Reference substance (positive control):
yes
Remarks:
Chloroacetamid
Key result
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
311 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.:
311 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
biomass deviation
Duration:
14 d
Dose descriptor:
LC50
Effect conc.:
>= 499 - <= 799 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
No mortality occured in both water and solvent controls. No mortality occured in the Terpineol multiconstituent treated units for tretment concentrations ranging between 121.1 and 311.1 mg/kg dw and the specimens appeared healthly. The LC50 was not calculated as the induced mortality exhibited a sudden increase from 2.5% up to 92.5% for the 499 and the 799 mg/kg dw treated groups respectively. Based on the regularity of the results within each treatment group, the LC50 value was considered to occur between 499 (% mortality < 20% in every case) and 799(% mortality> 80% in every case) mg/kg dw. Mean gain of biomass was 6.8% and 7.7% in the water and the solvent controls, respectively. Increase of biomass was the same level in the Terpineol multiconstituent treated groups for 121.1; 194.1 and 311.1 mg/kg concentrations.Mean weight was decreased in the 498.5 mg/kg treated group, especially because of replicate four where injured specimens were observed (-48.1%).
Results with reference substance (positive control):
As required, LC50 (14d) for chloroacetamid occured between 20 and 80 mg/kg. Injured specimens were observed in the 40 mg/kg treated group where mean percent mortality on day 14 was 42.5%. A 20mg/kg treatment resulted in biomass deviatioin of +5.5% similar to that of the control. Loss of biomass occured in most of the 40 mg/kg treated units where injured specimens were observed and % mortality was 42.5%.
Reported statistics and error estimates:
No data
Validity criteria fulfilled:
yes
Conclusions:
The test was considered as valid.
NOEC mortality (14d) = 311 mg/kg dry soil;
NOEC biomass deviation (14d) = 311 mg/kg dry soil
499 < LC50 (14 days)< 799 mg/kg dry soil
Executive summary:

The acute toxicity of Terpineol multiconstituent was determined on earthworm (Eisenia fetida) in a study performed according to Guideline OECD 207 including a GLP compliance statement. The organisms were exposed during 14 days using artificial soil substrate mixed with the test substance, the controls (water and solvent) or the reference substance (chloroacetamid). The monitored effects were the mortality and the loss of biomass. The pH and the moisture content were also monitored.

A range-finding test was performed initially and showed a LC50 (14d) between 100 and 1001 mg/kg dw. The nominal tested concentrations of the full test ranged between 121.1 and 798.9 mg/kg dry soil. Ten earthworms per test unit between 2 and 12 months and 4 replicates were used in the the full test.

No mortality occured in both water and solvent controls. No mortality occured in the units treated with Terpineol multiconstituent for concentrations ranging between 121.1 and 311.1 mg/kg dw and the specimens appeared healthly. The LC50 was not calculated as the induced mortality exhibited a sudden increase from 2.5% up to 92.5% for the 499 and the 799 mg/kg dw treated groups respectively. Based on the regularity of the results within each treatment group, the LC50 value was considered to occur between 499 and 799 mg/kg dry soil. As required, LC50 (14d) for chloroacetamid occured between 20 and 80 mg/kg.

The test was considered as valid. The NOEC mortality (14d) was 311 mg/kg dry soil. The NOEC biomass deviation (14d) is 311 mg/kg dry soil. The LC50 (14 days) was between 499 mg/kg dry soil and 799 mg/kg dry soil.

Description of key information

The acute toxicity of terpineol multiconstituent on earthworm (Eisenia fetida) was evaluated in a study performed according to Guideline OECD 207.

The organisms were exposed during 14 days using artificial soil substrate mixed with the test substance at concentrations between 121.1 mg/kg dry soil and 798.9 mg/kg dry soil.
NOEC (14d) = 311 mg/kg dry soil
499 < LC50 (14 days)< 799 mg/kg dry soil.

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:
499 mg/kg soil dw
Long-term EC10, LC10 or NOEC for soil macroorganisms:
311 mg/kg soil dw

Additional information