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Ecotoxicological information

Toxicity to soil macroorganisms except arthropods

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Administrative data

Endpoint:
toxicity to soil macroorganisms except arthropods: short-term
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
1980
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Study neither conducted according to internationally accepted guideline nor to GLP

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1980
Report date:
1980

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: Method described by Douvres et al., 1966
GLP compliance:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
N,N-dimethyldodecanamide
EC Number:
221-117-5
EC Name:
N,N-dimethyldodecanamide
Cas Number:
3007-53-2
Molecular formula:
C14H29NO
IUPAC Name:
N,N-dimethyldodecanamide
Details on test material:
- Name of the test substance: N,N-dimethyldodecanamide
- Purity: > 98 %

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
No concentrations were measured.

Test substrate

Vehicle:
yes
Details on preparation and application of test substrate:
TEST SOLUTIONS
- 0.1 % stock solution of each test compound in 95 % ethanol was prepared and sterilized by filtration (millipore 0.22 mµ) and stored at 5 °C until used in the vitro study.
- 0.1 % stock solution of thiabendazole (TBZ) in 0.1 N HCl was prepared and stored in the dark at room temperatire (22 °C)

PREPARATION OF THE TEST MEDIUM
- Test Medium: The appropiated concentrations of the test were added to the media RFN or API-1 by serially diluting the 0.1% stock solution of the test substance or TBZ with the respective medium.
- Additional substrate: Yes a pepsin supplement. The pepsin supplement was added to the medium API-1 after the required quantity of test compound or TBZ had been added to the medium.

Test organisms

Test organisms (species):
other: Ostertagia ostertagi
Animal group:
nematods
Details on test organisms:
TEST ORGANISMS
- Common Name: Ostertagia ostertagi

ACCLIMATATION
- Acclimatation conditions: Tap water at 5 °C
- Acclimatation period: 15 – 61 days

Study design

Study type:
laboratory study
Substrate type:
not specified
Limit test:
no
Total exposure duration:
35 d

Test conditions

Test temperature:
5 °C
pH:
7.3
Moisture:
No data
Details on test conditions:
TEST SOLUTIONS
- 0.1 % stock solution of each test compound in 95 % ethanol was prepared and sterilized by filtration (millipore 0.22 mµ) and stored at 5 °C until used in the vitro study.
- 0.1 % stock solution of thiabendazole (TBZ) in 0.1 N HCl was prepared and stored in the dark at room temperatire (22 °C)

PREPARATION OF THE TEST MEDIUM
- Test Medium: The appropiated concentrations of the test were added to the media RFN or API-1 by serially diluting the 0.1% stock solution of the test substance or TBZ with the respective medium.
- Addition of pepsin supplement: yes. The pepsin supplement was added to the medium API-1 after the required quantity of test compound or TBZ had been added to the medium.
- pH adjusted: yes. The test media RFN and API-1 + pepsin were adjusted to the required pH and used on the day of preparation.
- No. of organisms per container (treatment): 50 000
- No. of replicates per treatment group: 2- 4
- No. of replicates per control: 1
- No. of replicates per vehicle control: 1
- Test performance: The methods of Douvres et al. (1966) were used to transfer inocula to fresh media and new culture vessels, to test the sterility of all stocks of media, and to examine and evaluate larvae subjected to the 70-min exsheathing process and the larvae and adults that developed in 2-4 replicate cultures of 50 000 larvae each, in the two-step culture system.
- Preparation of the organisms for the exposure: Suspensions of 50 000 infective larvae each, that had been stored from 15 to 61 days in tap water at 5°C, were cleaned and suspended in medium RFN containing the appropriate concentration (0.25-5.0 p.p.m.) of the test compounds and then subjected to the exsheathing process (Douvres and Malakatis, 1977). These larvae were suspended in 50 mL of medium RFN with test compound in roller bottles and were used as the inoculum in the two-step culture system. The inoculum was transferred to the culture system (Step 1) which consisted of medium RFN with test compound at pH 7.3, in a gas phase of 95% air/5% carbon dioxide, and the culture was incubated for 3 days at 39°C. On Day 3, the larvae were transferred to medium API-1 + pepsin (Step 2) with test compound at pH 4.5 in a gas phase of 85% nitrogen/5% oxygen/ 10% carbon dioxide (Air Products, Allentown, PA) and kept at pH 4.5 for 6 or 7 days, and thereafter at a pH of 6.0. A control (non-treated media) culture system was also prepared.

EFFECT PARAMETERS MEASURED : Toxicity effects (decreased motility or paralysis; reduced survival; delayed or blocked third or fourth ecdysis; lowered yields of advanced stages; decreased production of fertile and non fertile eggs on Ostertagia ostertagi after 3, 17 and 35 days.

EVALUATION OF THE RESULTS
Evaluation of the cultures involved the use of data without regard to number of larvae or adults to determine the most advanced stage of development attained and the earliest attainment of an advanced stage, and the use of data based on averages of estimated percentages or actual counts of larvae and adults to determine survival and yield of advanced stages. Developmental stages of the nematode were considered moribund or dead by criteria described previously (Douvres et al., 1966). The cultivation of cultures included one or two controls. Cultures were judged free of contamination if there was no visible evidence of fungi or bacteria.

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Nominal concnetrations: 0 (control), 1, 2.5 and 5 mg/L
- Measured concentrations: No concentrations were measured.

- Results used to determine the conditions for the definitive study:

Nominal and measured concentrations:
Nominal concentrations: 0.25 – 5 mg/L
Measured concentration; No concentrations were measured
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 d
Dose descriptor:
EC100
Effect conc.:
> 5 other: ppm
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95 % C.L. not determined
Duration:
17 d
Dose descriptor:
EC50
Effect conc.:
> 2.5 - < 5 other: ppm
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95 % C.L not determined
Duration:
35 d
Dose descriptor:
EC50
Effect conc.:
> 2.5 - < 5 other: ppm
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 35 % C.L. not determined
Details on results:
In control Step 1 cultures of medium RFN, 98-100% of O. ostertagi infective larvae had exsheathed by Day 1 and development to third stage (L3) started on Day 2. By Day 3, 75-100% of the larvae had developed to the L3 stage. The larvae were normal and their motility was vigorous. The ranges of concentrations of the test substance required to inhibit development or kill 100% of the larvae after 3 days in culture medium RFN were greater than 5 p.p.m.. The test substance at a concentration of 1.0 p.p.m. permitted development of larvae to egg-laying adults. The EC100 was determined to be greater than 5 ppm. The 17d-NOEC and the 35 d- NOEC were determined to be lower than 1 ppm, the 17d-EC50 was determined to be in a range between 2.5 and 5 ppm and the 35d - EC50 was determined to be greater than 2.5 ppm.
Results with reference substance (positive control):
No results with reference substance as positive control were determined.
Reported statistics and error estimates:
No reported statistics and error were estimated.

Applicant's summary and conclusion

Conclusions:
The objective of this study was to determine the toxic effects of the test substance on Ostertagia ostertagi after 3, 17 and 35 days. The 35d-NOEC was determined to be lower than 1 ppm and the 17d-EC50 was determined to be in a range between 2.5 and 5 ppm and the 35d-EC50 was determined to be greater than 2.5 ppm.
Executive summary:

The objective of this study was to determine the concentrations of the test substance required to inhibited development or kill 100 % of infective larvae of Ostertagia ostertagi and to determine the toxic effects of the test substance on survival and yield of advanced stages of Ostertagia ostertagi, developed from infective larvae in a two-step roller culture system after 3, 17 and 35 days. Suspensions of 50 000 infective larvae each, that had been stored from 15 to 61 days in tap water at 5°C, were cleaned and suspended in medium RFN containing the appropriate concentration (0.25--5.0 p.p.m.) of the test compounds and then subjected to the exsheathing process (Douvres and Malakatis, 1977). These larvae were suspended in 50 mL of medium RFN with test compound in roller bottles and were used as the inoculum in the two-step culture system. The inoculum was transferred to the culture system (Step 1) which consisted of medium RFN with test compound at pH 7.3, in a gas phase of 95% air/5% carbon dioxide, and the culture was incubated for 3 days at 39°C. On Day 3, the larvae were transferred to medium API-1 + pepsin (Step 2) with test compound at pH 4.5 in a gas phase of 85% nitrogen/5% oxygen/ 10% carbon dioxide (Air Products, Allentown, PA) and kept at pH 4.5 for 6 or 7 days, and thereafter at a pH of 6.0. A control (non-treated media) culture system was also prepared. In control Step 1 cultures of medium RFN, 98-100% of O. ostertagi infective larvae had exsheathed by Day 1 and development to third stage (L3) started on Day 2. By Day 3, 75--100% of the larvae had developed to the L3 stage. The larvae were normal and their motility was vigorous. The ranges of concentrations of the test substance required to inhibit development or kill 100% of the larvae after 3 days in culture medium RFN were greater than 5 p.p.m. The test substance at a concentration of 1.0 p.p.m. permitted development of larvae to egg-laying adults. The 3d-EC100 was determined to be greater than 5 p.p.m.. The 17d-NOEC and the 35 d- NOEC were determined to be lower than 1 p.p.m., the 17d-EC50 was determined to be in a range between 2.5 and 5 ppm. and the 35d - EC50 was determined to be greater than 2.5 ppm.