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Ecotoxicological information

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:
23 August 1996 – 18 August 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to GLP requirements. Study carried out according to ISO 11268
Qualifier:
according to
Guideline:
ISO 11268-2 (Effects of Pollutants on Earthworms. 2. Determination of Effects on Reproduction)
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
yes
Details on preparation and application of test substrate:
AHTN was dissolved in acetone. The AHTN solution was mixed with quarz sand. The acetone was evaporated and the quarz sand (containing AHTN) was mixed with the artificial soil.
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
The study was performed with earthworm Eisenia fetida bred under standardised conditions (see OECD 207) by IBACON (range finding test) and Kraut & Rüben, Doris Haber, Zeilstrasse 40, D-64367 Mühltal (main test).
Adult earthworms with well developed clitellum of an age between 8 and 9 month (range finding test) and between 9 and 10 month (main test) were used. The body weights of the earthworms in the main test were 342 - 540 mg, but did not differ for more than 100 mg within this range in each test container. Prior to test initiation, the earthworms were acclimated in artificial soil under the test conditions for a period of 4 days. In this time the earthworms were fed from the same food source as in the definitive test.
The eartworms were weekly fed with cattle manure.
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
8 wk
Remarks:
after 4 weeks the adult worms were removed and the offspring produced stayed in the test containers for another four weeks
Test temperature:
The test containers were exposed in a ventilated room at 20 ± 3 °C.
pH:
At the beginning of the study, the pH of the artificial soil was between 6.3 and 6.4. At the end of the study the pH of the artificial soil was in a range of 6.2 and 6.3. These values were within the validity criteria of the guidelines (6.0 ± 0.5).
Moisture:
The water content was between 33.9 % and 34.4 % (of dry weight) at start of the study and in a range from 37.8.% to 42.0 % (of dry weight) at the end of the study.
Reference substance (positive control):
yes
Remarks:
Benomyl, Purity: 50 %, Batch: MAR94D21 1031402, applied amount in this Study: 15 mg per kg artificial soil (dry weight).
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
105 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
and food consumption
Duration:
56 d
Dose descriptor:
LOEC
Effect conc.:
250 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
and food consumption
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 250 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth
Remarks:
and mortality of adults
Details on results:
Mortality and body weight (after 4 weeks)
The results of this study did not reveal any acute toxic effect. None of the adult earthworms died within the 4 weeks of experimental time. The body weights of the earthworms increased compared to the control slightly in all treatment groups except the reference compound group. Here a not significant loss of body weight was measured (Dunnetts test p = 0.05).
No behavioural abnormalities were observed for any treatments.

Reproduction (after 8 weeks)
Reproduction of the earthworms exposed to AHTN was slightly, but not significantly reduced in the 105 mg/kg treatment group. In contrast reproduction of the earthworms exposed to 250 mg/kg AHTN was significantly reduced. There was a statistically significant reduction of reproduction in the Reference compound group (Dunnetts test p = 0.05).
These results prove that AHTN does affect reproduction if applied in concentrations up to 105 mg/kg.

Food consumption
Applications of up to 105 mg/kg of AHTN do not lead to a reduction of food consumption.

Details on results are displayed in table 1.
Results with reference substance (positive control):
Benomyl clearly showed an effect on body weight increase. The body weight of the worms exposed to Benomyl actually decreased, while the other worms showed an increase in weight. Reproduction was also clearly significantly different from the control and the worms exposed to AHTN.
Reported statistics and error estimates:
For each concentration a statistical analysis of the homogeneity and normality of replicate results was made. No significant difference (Dunnetts test p = 0.05) between the water treated control and the solvent control was observed. Appropriate statistical analysis was performed to determine whether differences between the control and the test concentrations are significant (Dunnetts test p = 0.05).

Table 1 Body weight differences, reproduction rates and food consumption against the concentration (including controls and reference).

Body Weight Differences Reproduction Food Consumption
Mean SD Mean Mean SD Mean SD
mg/worm % no of worms g added food
Control 9 ± 46 2 238 ± 21 21.2 ± 1.1
Solvent
Control
32 ± 21 7.6 244 ± 31 21.2 ± 0.2
8 20 ± 56 4.7 216 ± 60 20.4 ± 0.8
19 55 ± 42 13 259 ± 35 21.6 ± 1.1
45 69 ± 46 16.4 230 ± 36 21.6 ± 0.6
105 62 ± 38 15.5 206 ± 29 21.3 ± 0.6
250 38 ± 49 8.4 94 ± 44 18.2 ± 1.8
Reference
compound
-45 ± 25 -10.2 3 ± 3 16.2 ± 1.4
Validity criteria fulfilled:
yes
Remarks:
Adult mortality in the controls was ≤10 %. Biomass loss in the controls did not exceed 20 %. Effects on reproduction (p = 0.05) in the reference substance was observed.
Conclusions:
In a study according to ISO 11268 carried out under GLP (Rel.1), survival and body weights of adult Eisenia fetida were not affected after 28 days of exposure up to and including 250 mg AHTN/kg artificial soil dw (highest concentration tested). After 56 days the food consumption and reproduction were shown to be reduced by exposure to a concentration of 250 mg/kg. AHTN causes sub-lethal effects at a concentration of 250 mg/kg. The NOEC was 105 mg/kg and the LOEC 250 mg/kg.
Executive summary:

The effects on survival, body weight and reproduction of the adult earthworm Eisenia fetida, on the survival of the descendants and on the approximated food consumption of the earthworms were investigated in a eight weeks study according to ISO 11268 carried out under GLP (Rel. 1). The worms were exposed in artificial soil containing 10% of dry organic matter. Nominal concentrations in soil were 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 was left in the test containers for another four weeks. No mortality or growth inhibition of the adults was observed after 4 weeks in concentrations up to and including 250 mg/kg. In the range finding test 100% mortality occurred after 14 days exposure to 1000 mg/kg. Reproduction was not significantly affected up to concentrations of 105 mg/kg (14% inhibition – not statistically significant, NOEC). At the level of the LOEC (250 mg/kg), the reproduction was 39% of the control.

Description of key information

The NOEC was determined by exposing earthworms to AHTN in artificial soil. The effects of the test item on mortality, reproduction, food consumption and growth were assessed. The NOEC was 105 mg/kg with for reproduction as the most sensitive effect. The NOEC derived from the study was normalised to standard soil (based on organic material). Consequently, the OC-normalised NOEC is 105 / 10% x 3.4% = 36 mg/kg.

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for soil macroorganisms:
36 mg/kg soil dw

Additional information

The NOEC of 105 mg/kg soil dw was determined by exposing earthworms to the test item in artificial soil. The lowest NOEC was found for reproduction and food consumption.

The soil in the test contains 10% dry organic material (Sphagnum peat) whereas the standard soil of the Technical Guidance Document contains 3.4% organic material. Consequently, the OC -normalised NOEC is 105 / 10% x 3.4% = 36 mg/kg.